bluuebunny/crossref_metadata_2025 huggingface dataset:
Roles of the
Escherichia coli
RecA Protein and the Global SOS Response in Effecting DNA Polymerase Selection In Vivo
Authors: Maul Robert W. , Sutton Mark D.
Published: November 2005

The
Escherichia coli
β sliding clamp protein is proposed to play an important role in effecting switches between different DNA polymerases during replication, repair, and translesion DNA synthesis. We recently described how strains bearing the
dnaN159
allele, which encodes a mutant form of the β clamp (β159), display a UV-sensitive phenotype that is suppressed by inactivation of DNA polymerase IV (M. D. Sutton, J. Bacteriol. 186:6738-6748, 2004). As part of an ongoing effort to understand mechanisms of DNA polymerase management in
E. coli
, we have further characterized effects of the
dnaN159
allele on polymerase usage. Three of the five
E.coli
DNA polymerases (II, IV, and V) are regulated as part of the global SOS response. Our results indicate that elevated expression of the
dinB
-encoded polymerase IV is sufficient to result in conditional lethality of the
dnaN159
strain. In contrast, chronically activated RecA protein, expressed from the
recA730
allele, is lethal to the
dnaN159
strain, and this lethality is suppressed by mutations that either mitigate RecA730 activity (i.e., Δ
recR
), or impair the activities of DNA polymerase II or DNA polymerase V (i.e., Δ
polB
or Δ
umuDC
). Thus, we have identified distinct genetic requirements whereby each of the three different SOS-regulated DNA polymerases are able to confer lethality upon the
dnaN159
strain, suggesting the presence of multiple mechanisms by which the actions of the cell's different DNA polymerases are managed in vivo.
Bacillus subtilis
Cysteine Synthetase Is a Global Regulator of the Expression of Genes Involved in Sulfur Assimilation
Authors: Albanesi Daniela , Mansilla Maria Cecilia , Schujman Gustavo E. , de Mendoza Diego
Published: November 2005

The synthesis of
l
-cysteine, the major mechanism by which sulfur is incorporated into organic compounds in microorganisms, occupies a significant fraction of bacterial metabolism. In
Bacillus subtilis
the
cysH
operon, encoding several proteins involved in cysteine biosynthesis, is induced by sulfur starvation and tightly repressed by cysteine. We show that a null mutation in the
cysK
gene encoding an
O
-acetylserine-(thiol)lyase, the enzyme that catalyzes the final step in cysteine biosynthesis, results in constitutive expression of the
cysH
operon. Using DNA microarrays we found that, in addition to
cysH
, almost all of the genes required for sulfate assimilation are constitutively expressed in
cysK
mutants. These results indicate that CysK, besides its enzymatic role in cysteine biosynthesis, is a global negative regulator of genes involved in sulfur metabolism.
Functional Analysis of All Aminotransferase Proteins Inferred from the Genome Sequence ofCorynebacterium glutamicum
Authors: Marienhagen Jan , Kennerknecht Nicole , Sahm Hermann , Eggeling Lothar
Published: November 2005

Twenty putative aminotransferase (AT) proteins ofCorynebacterium glutamicum, or rather pyridoxal-5′-phosphate (PLP)-dependent enzymes, were isolated and assayed among others withl-glutamate,l-aspartate, andl-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference)l-glutamate > 2-aminobutyrate >l-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizesl-alanine to aminate 2-oxo-isovalerate, thel-valine precursor, and 2-oxo-butyrate. A second AT active with thel-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs forl-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far.
The Twin-Arginine Translocation Pathway of
Mycobacterium smegmatis
Is Functional and Required for the Export of Mycobacterial β-Lactamases
Authors: McDonough Justin A. , Hacker Kari E. , Flores Anthony R. , Pavelka Martin S. , Braunstein Miriam
Published: November 2005

The twin-arginine translocation (Tat) pathway exports folded proteins across the bacterial cytoplasmic membrane and is responsible for the proper extracytoplasmic localization of proteins involved in a variety of cellular functions, including pathogenesis. The
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
genomes contain open reading frames with homology to components of the Tat export system (TatABC) as well as potential Tat-exported proteins possessing N-terminal signal sequences with the characteristic twin-arginine motif. Due to the importance of exported virulence factors in the pathogenesis of
M. tuberculosis
and the limited understanding of mycobacterial protein export systems, we sought to determine the functional nature of the Tat export pathway in mycobacteria. Here we describe phenotypic analyses of Δ
tatA
and Δ
tatC
deletion mutants of
M. smegmatis
, which demonstrated that
tatA
and
tatC
encode components of a functional Tat system capable of exporting characteristic Tat substrates. Both mutants displayed a growth defect on agar medium and hypersensitivity to sodium dodecyl sulfate. The mutants were also defective in the export of active β-lactamases of
M. smegmatis
(BlaS) and
M. tuberculosis
(BlaC), both of which possess twin-arginine signal sequences. The Tat-dependent nature of BlaC was further revealed by mutation of the twin-arginine motif. Finally, we demonstrated that replacement of the native signal sequence of BlaC with the predicted Tat signal sequences of
M. tuberculosis
phospholipase C proteins (PlcA and PlcB) resulted in the Tat-dependent export of an enzymatically active ′BlaC. Thus, ′BlaC can be used as a genetic reporter for Tat-dependent export in mycobacteria.
Evidence for Acquisition ofLegionellaType IV Secretion Substrates via Interdomain Horizontal Gene Transfer
Authors: de Felipe Karim Suwwan , Pampou Sergey , Jovanovic Oliver S. , Pericone Christopher D. , Ye Senna F. , Kalachikov Sergey , Shuman Howard A.
Published: November 2005

Intracellular pathogens exploit host cell functions to create a replication niche inside eukaryotic cells. The causative agent of Legionnaires' disease, the γ-proteobacteriumLegionella pneumophila, resides and replicates within a modified vacuole of protozoan and mammalian cells.L. pneumophilatranslocates effector proteins into host cells through the Icm-Dot complex, a specialized type IVB secretion system that is required for intracellular growth. To find out if some effector proteins may have been acquired through interdomain horizontal gene transfer (HGT), we performed a bioinformatic screen that searched for eukaryotic motifs in all open reading frames of theL. pneumophilaPhiladelphia-1 genome. We found 44 uncharacterized genes with many distinct eukaryotic motifs. Most of these genes contain G+C biases compared to otherL. pneumophilagenes, supporting the theory that they were acquired through HGT. Furthermore, we found that several of them are expressed and up-regulated in stationary phase in an RpoS-dependent manner. In addition, at least seven of these gene products are translocated into host cells via the Icm-Dot complex, confirming their role in the intracellular environment. Reminiscent of the case with most Icm-Dot substrates, most of the strains containing mutations in these genes grew comparably to the parent strain intracellularly. Our findings suggest that inL. pneumophila, interdomain HGT may have been a major mechanism for the acquisition of determinants of infection.
Mapping of a YscY Binding Domain within the LcrH Chaperone That Is Required for Regulation of
Yersinia
Type III Secretion
Authors: Bröms Jeanette E. , Edqvist Petra J. , Carlsson Katrin E. , Forsberg Åke , Francis Matthew S.
Published: November 2005

Type III secretion systems are used by many animal and plant interacting bacteria to colonize their host. These systems are often composed of at least 40 genes, making their temporal and spatial regulation very complex. Some type III chaperones of the translocator class are important regulatory molecules, such as the LcrH chaperone of
Yersinia pseudotuberculosis
. In contrast, the highly homologous PcrH chaperone has no regulatory effect in native
Pseudomonas aeruginosa
or when produced in
Yersinia.
In this study, we used LcrH-PcrH chaperone hybrids to identify a discrete region in the N terminus of LcrH that is necessary for YscY binding and regulatory control of the
Yersinia
type III secretion machinery. PcrH was unable to bind YscY and the homologue Pcr4 of
P. aeruginosa.
YscY and Pcr4 were both essential for type III secretion and reciprocally bound to both substrates YscX of
Yersinia
and Pcr3 of
P. aeruginosa.
Still, Pcr4 was unable to complement a Δ
yscY
null mutant defective for type III secretion and
yop-
regulatory control in
Yersinia
, despite the ability of YscY to function in
P. aeruginosa.
Taken together, we conclude that the cross-talk between the LcrH and YscY components represents a strategic regulatory pathway specific to
Yersinia
type III secretion.
CapE, a 47-Amino-Acid Peptide, Is Necessary for
Bacillus anthracis
Polyglutamate Capsule Synthesis
Authors: Candela Thomas , Mock Michèle , Fouet Agnès
Published: November 2005

Polyglutamate is found in various bacteria, but displays different functions depending on the species and their environment. Here, we describe a minimal polyglutamate synthesis system in
Bacillus anthracis
. In addition to the three genes previously described as sufficient for polyglutamate synthesis, this system includes a small open reading frame,
capE
, belonging to the
cap
operon. The polyglutamate system's requirement for the five
cap
genes, for capsulation and anchoring, was assayed in nonpolar mutants. The
capA
,
capB
,
capC
, and
capE
genes are all necessary and are sufficient for polyglutamate synthesis by
B. anthracis. capD
is required for polyglutamate anchoring to the peptidoglycan. The 47-amino-acid peptide encoded by
capE
is localized in the
B. anthracis
membrane. It is not a regulator and it is required for polyglutamate synthesis, suggesting that it has a structural role in polyglutamate synthesis. CapE appears to interact with CapA.
Bacillus subtilis ywtC
is similar to
capE
and we named it
pgsE
. Genes similar to
capE
or
pgsE
were found in
B. subtilis
natto,
Bacillus licheniformis
, and
Staphylococcus epidermidis
, species that produce polyglutamate. All the bacterial polyglutamate synthesis systems analyzed show a similar genetic organization and, we suggest, the same protein requirements.
Characterization of
Mycobacterium tuberculosis
Rv3676 (CRP
Mt
), a Cyclic AMP Receptor Protein-Like DNA Binding Protein
Authors: Bai Guangchun , McCue Lee Ann , McDonough Kathleen A.
Published: November 2005

Little is known about cyclic AMP (cAMP) function in
Mycobacterium tuberculosis
, despite its ability to encode 15 adenylate cyclases and 10 cNMP-binding proteins.
M. tuberculosis
Rv3676, which we have designated CRP
Mt
, is predicted to be a cAMP-dependent transcription factor. In this study, we characterized CRP
Mt
's interactions with DNA and cAMP, using experimental and computational approaches. We used Gibbs sampling to define a CRP
Mt
DNA motif that resembles the cAMP receptor protein (CRP) binding motif model for
Escherichia coli
. CRP
Mt
binding sites were identified in a total of 73 promoter regions regulating 114 genes in the
M. tuberculosis
genome, which are being explored as a regulon. Specific CRP
Mt
binding caused DNA bending, and the substitution of highly conserved nucleotides in the binding site resulted in a complete loss of binding to CRP
Mt
. cAMP enhanced CRP
Mt
's ability to bind DNA and caused allosteric alterations in CRP
Mt
conformation. These results provide the first direct evidence for cAMP binding to a transcription factor in
M. tuberculosis
, suggesting a role for cAMP signal transduction in
M. tuberculosis
and implicating CRP
Mt
as a cAMP-responsive global regulator.
Alternate Sigma Factor RpoS Is Required for the In Vivo-Specific Repression ofBorrelia burgdorferiPlasmid lp54-BorneospAandlp6.6Genes
Authors: Caimano Melissa J. , Eggers Christian H. , Gonzalez Cynthia A. , Radolf Justin D.
Published: November 2005

While numerous positively regulated loci have been characterized during the enzootic cycle ofBorrelia burgdorferi, very little is known about the mechanism(s) involved in the repression of borrelial loci either during tick feeding or within the mammalian host. Here, we report that the alternative sigma factor RpoS is required for the in vivo-specific repression of at least two RpoD-dependentB. burgdorferiloci,ospAandlp6.6. The downregulation ofospAandIp6.6appears to require either a repressor molecule whose expression is RpoS dependent or an accessory factor which enables RpoS to directly interact with theospAandIp6.6promoter elements, thereby blocking transcription by RpoD. The central role for RpoS during the earliest stages of host adaptation suggests that tick feeding imparts signals to spirochetes that trigger the RpoS-dependent repression, as well as expression, of in vivo-specific virulence factors critical for the tick-to-mammalian host transition.
Tryptophan Catabolism: Identification and Characterization of a New Degradative Pathway
Authors: Colabroy Keri L. , Begley Tadhg P.
Published: November 2005

A new tryptophan catabolic pathway is characterized from
Burkholderia cepacia
J2315. In this pathway, tryptophan is converted to 2-amino-3-carboxymuconate semialdehyde, which is enzymatically degraded to pyruvate and acetate via the intermediates 2-aminomuconate and 4-oxalocrotonate. This pathway differs from the proposed mammalian pathway which converts 2-aminomuconate to 2-ketoadipate and, ultimately, glutaryl-coenzyme A.
Cadmium inhibits both intrinsic and extrinsic apoptotic pathways in renal mesangial cells
Authors: Gunawardana C. Geeth , Martinez Raul E. , Xiao Weiqun , Templeton Douglas M.
Published: November 2005

Cadmium is a potent nephrotoxin that has been shown to induce apoptosis in some cells but also to prevent it under certain circumstances. In several clinical situations and experimental models of injury to the renal glomerulus, pathological proliferation of mesangial cells is followed by resolution involving mesangial cell apoptosis. We investigated the effects of Cd2+ on rat mesangial cells induced to undergo apoptosis through either the extrinsic receptor-mediated pathway or the intrinsic mitochondrial-dependent pathway. Camptothecin initiated the intrinsic pathway with activation of caspase-9 and caspase-dependent cleavage of procaspase-3. Tumor necrosis factor-α (TNF-α) initiated caspase-8 activity and cleavage of pro-caspase-3 at the convergence point of the two pathways. However, pro-caspase-8 levels were low, and caspase-9 was also activated in response to TNF-α, characteristic of what have been termed type II cells. With both TNF-α and camptothecin, concurrent exposure to 10 μM CdCl2 suppressed DNA laddering, nuclear condensation, and pro-caspase-3 cleavage. It also decreased activity of both caspase-8 and caspase-9, prevented caspase-8-dependent cleavage of the proapoptotic factor Bid, and suppressed release of cytochrome c from mitochondria. At this 10-μM concentration, Cd2+ was unique among a number of metal ions in preventing DNA fragmentation. We conclude that Cd2+ is anti-apoptotic in rat mesangial cells, acting by a mechanism that may involve general caspase inhibition. This may have consequences for the resolution of nephritis in situations of mesangial cell hyperproliferation.
Intermittent Visuomotor Processing in the Human Cerebellum, Parietal Cortex, and Premotor Cortex
Authors: Vaillancourt David E. , Mayka Mary A. , Corcos Daniel M.
Published: November 2005

The cerebellum, parietal cortex, and premotor cortex are integral to visuomotor processing. The parameters of visual information that modulate their role in visuomotor control are less clear. From motor psychophysics, the relation between the frequency of visual feedback and force variability has been identified as nonlinear. Thus we hypothesized that visual feedback frequency will differentially modulate the neural activation in the cerebellum, parietal cortex, and premotor cortex related to visuomotor processing. We used functional magnetic resonance imaging at 3 Tesla to examine visually guided grip force control under frequent and infrequent visual feedback conditions. Control conditions with intermittent visual feedback alone and a control force condition without visual feedback were examined. As expected, force variability was reduced in the frequent compared with the infrequent condition. Three novel findings were identified. First, infrequent (0.4 Hz) visual feedback did not result in visuomotor activation in lateral cerebellum (lobule VI/Crus I), whereas frequent (25 Hz) intermittent visual feedback did. This is in contrast to the anterior intermediate cerebellum (lobule V/VI), which was consistently active across all force conditions compared with rest. Second, confirming previous observations, the parietal and premotor cortices were active during grip force with frequent visual feedback. The novel finding was that the parietal and premotor cortex were also active during grip force with infrequent visual feedback. Third, right inferior parietal lobule, dorsal premotor cortex, and ventral premotor cortex had greater activation in the frequent compared with the infrequent grip force condition. These findings demonstrate that the frequency of visual information reduces motor error and differentially modulates the neural activation related to visuomotor processing in the cerebellum, parietal cortex, and premotor cortex.
Neonatal Cortical Ablation Disrupts Multisensory Development in Superior Colliculus
Authors: Jiang Wan , Jiang Huai , Stein Barry E.
Published: November 2005

The ability of cat superior colliculus (SC) neurons to synthesize information from different senses depends on influences from two areas of the cortex: the anterior ectosylvian sulcus (AES) and the rostral lateral suprasylvian sulcus (rLS). Reversibly deactivating the inputs to the SC from either of these areas in normal adults severely compromises this ability and the SC-mediated behaviors that depend on it. In this study, we found that removal of these areas in neonatal animals precluded the normal development of multisensory SC processes. At maturity there was a substantial decrease in the incidence of multisensory neurons, and those multisensory neurons that did develop were highly abnormal. Their cross-modal receptive field register was severely compromised, as was their ability to integrate cross-modal stimuli. Apparently, despite the impressive plasticity of the neonatal brain, it cannot compensate for the early loss of these cortices. Surprisingly, however, neonatal removal of either AES or rLS had comparatively minor consequences on these properties. At maturity multisensory SC neurons were quite common: they developed the characteristic spatial register among their unisensory receptive fields and exhibited normal adult-like multisensory integration. These observations suggest that during early ontogeny, when the multisensory properties of SC neurons are being crafted, AES and rLS may have the ability to compensate for the loss of one another's cortico-collicular influences so that normal multisensory processes can develop in the SC.
Rac3-Mediated Transformation Requires Multiple Effector Pathways
Authors: Keller Patricia J. , Gable Christyn M. , Wing Michele R. , Cox Adrienne D.
Published: November 2005

Our initial characterization of Rac3, a close relative of the small GTPase Rac1, established its ability to promote membrane ruffling, transformation, and activation of c-jun transcriptional activity. The finding that Rac3 is transforming, and its similarity to Rac1, a protein that has a well-established connection to many processes important for cancer progression, prompted further investigation into Rac3 transformation. We used effector domain mutants (EDMs) to explore the relationship among Rac signaling, transformation, and effector usage. All Rac3 EDMs tested (N26D, F37L, Y40C, and N43D) retained the ability to promote membrane ruffling and focus formation. In contrast, only the N43D mutant promoted anchorage independence. This differs from Rac1, where both N26D and N43D mutants were impaired in both types of transformation. To learn more about the signaling pathways involved, we did luciferase reporter assays and glutathione S-transferase pull-down assays for effector binding. We found evidence for a functional link between activation of phospholipase Cβ2 by Rac3 and signaling to the serum response factor (SRF). Surprisingly, we also found that Rac3 binds poorly to the known Rac1 effectors mixed lineage kinases 2 and 3 (MLK2 and MLK3). Transcription of cyclin D1 was the only pathway that correlated with growth in soft agar. Our experiments show that activation of membrane ruffling and transcriptional activation of c-jun, SRF, or E2F are not sufficient to promote anchorage-independent growth mediated by Rac3. Instead, multiple effector pathways are required for Rac3 transformation, and these overlap partially but not completely with those used by Rac1.
Quantification of Cellular Proliferation in Tumor and Normal Tissues of Patients with Breast Cancer by [18F]Fluorothymidine-Positron Emission Tomography Imaging: Evaluation of Analytical Methods
Authors: Kenny Laura M. , Vigushin David M. , Al-Nahhas Adil , Osman Safiye , Luthra Sajinder K. , Shousha Sami , Coombes R. Charles , Aboagye Eric O.
Published: November 2005

There is an unmet need to develop imaging methods for the early and objective assessment of breast tumors to therapy. 3′-Deoxy-3′-[18F]fluorothymidine ([18F]FLT)–positron emission tomography represents a new approach to imaging thymidine kinase activity, and hence, cellular proliferation. We compared graphical, spectral, and semiquantitative analytic methodologies for quantifying [18F]FLT kinetics in tumor and normal tissue of patients with locally advanced and metastatic breast cancer. The resultant kinetic parameters were correlated with the Ki-67 labeling index from tumor biopsies. [18F]FLT accumulation was detected in primary tumor, nodal disease, and lung metastasis. In large tumors, there was substantial heterogeneity in regional radiotracer uptake, reflecting heterogeneity in cellular proliferation; radiotracer uptake in primary tumors also differed from that of metastases. [18F]FLT was metabolized in patients to a single metabolite [18F]FLT-glucuronide. Unmetabolized [18F]FLT accounted for 71.54 ± 1.50% of plasma radioactivity by 90 minutes. The rate constant for the metabolite-corrected net irreversible uptake of [18F]FLT (Ki) ranged from 0.6 to 10.4 × 10−4 and from 0 to 0.6 × 10−4 mL plasma cleared/s/mL tissue in tumor (29 regions, 15 patients) and normal tissues, respectively. Tumor Ki and fractional retention of radiotracer determined by spectral analysis correlated with Ki-67 labeling index (r = 0.92, P < 0.0001 and r = 0.92, P < 0.0001, respectively). These correlations were superior to those determined by semiquantitative methods. We conclude that [18F]FLT-positron emission tomography is a promising clinical tool for imaging cellular proliferation in breast cancer, and is most predictive when analyzed by graphical and spectral methods.
Renal Cancer Treatment with Low Levels of Mixed Chimerism Induced by Nonmyeloablative Regimen Using Cyclophosphamide in Mice
Authors: Harano Masahiko , Eto Masatoshi , Iwai Toshiro , Tatsugami Katsunori , Kiyoshima Keijiro , Kamiryo Yoriyuki , Tsuneyoshi Masazumi , Yoshikai Yasunobu , Naito Seiji
Published: November 2005

Recently, much attention has been paid to nonmyeloablative allogeneic stem cell transplantation for the treatment of metastatic renal cancer. Mature donor T cells cause graft-versus-host disease (GVHD) although they are also the main mediators of the beneficial graft-versus-tumor activity associated with this treatment. Hence, the segregation of the graft-versus-tumor activity from GVHD is an important challenge in managing the clinical course of treatment. We previously reported a series of studies regarding the allograft tolerance induced by allogeneic spleen cells (with bone marrow cells) and cyclophosphamide in mice. Here, we show a modified cyclophosphamide-induced tolerance system for the treatment of murine renal cell carcinoma, RENCA, by shifting the equal balance between graft-versus-host and host-versus-graft reactions toward graft-versus-host reaction with donor lymphocyte infusion. Our results clearly show the antitumor activity against RENCA with only low levels of mixed chimerism in the periphery and the in vivo and in vitro acquired immunity against RENCA even when mixed chimerism is almost undetectable. Because the withdrawal of mixed chimerism reduces the risk of GVHD, the antitumor activity is thus sequentially segregated from the initial GVHD in our model. We believe that this is the first unique model system of nonmyeloablative allogeneic hemopoietic cell transplantation to ever be reported for the treatment of renal cancer.
Progestins Initiate a Luminal to Myoepithelial Switch in Estrogen-Dependent Human Breast Tumors without Altering Growth
Authors: Sartorius Carol A. , Harvell Djuana M.E. , Shen Tianjie , Horwitz Kathryn B.
Published: November 2005

Although long-term clinical use of progestins is associated with an increased incidence of breast cancers, their role in established cancers is unclear. Estrogens are considered to be the main mitogens in the majority of breast cancers. Whether progesterone affects proliferation and/or differentiation is under debate. To assess the role of progesterone in established breast cancers, we used T47D human breast cancer cells that are estrogen receptor (ER) positive and either progesterone receptor (PR) negative or positive for PRA, PRB, or both. These cells were grown as strictly estrogen-dependent solid tumors in ovariectomized female nude mice. Progesterone or medroxyprogesterone acetate (MPA) alone did not support tumor growth, nor did progesterone or MPA given simultaneously with estrogen significantly alter estrogen-dependent tumor growth. However, treatment of mice bearing ER+PR+ but not ER+PR− tumors with either progesterone or MPA increased expression of the myoepithelial cytokeratins (CK) 5 and 6 in a subpopulation of tumor cells. These CK5+/CK6+ cells had decreased expression of luminal epithelial CK8, CK18, and CK19. We conclude that progestins exert differentiative effects on tumors characterized by transition of a cell subpopulation from luminal to myoepithelial. This may not be beneficial, however, because such a phenotype is associated with poor prognosis.
Resveratrol Inhibits Myeloma Cell Growth, Prevents Osteoclast Formation, and Promotes Osteoblast Differentiation
Authors: Boissy Patrice , Andersen Thomas L. , Abdallah Basem M. , Kassem Moustapha , Plesner Torben , Delaissé Jean-Marie
Published: November 2005

Multiple myeloma is characterized by the accumulation of clonal malignant plasma cells in the bone marrow, which stimulates bone destruction by osteoclasts and reduces bone formation by osteoblasts. In turn, the changed bone microenvironment sustains survival of myeloma cells. Therefore, a challenge for treating multiple myeloma is discovering drugs targeting not only myeloma cells but also osteoclasts and osteoblasts. Because resveratrol (trans-3,4′,5-trihydroxystilbene) is reported to display antitumor activities on a variety of human cancer cells, we investigated the effects of this natural compound on myeloma and bone cells. We found that resveratrol reduces dose-dependently the growth of myeloma cell lines (RPMI 8226 and OPM-2) by a mechanism involving cell apoptosis. In cultures of human primary monocytes, resveratrol inhibits dose-dependently receptor activator of nuclear factor-κB (NF-κB) ligand–induced formation of tartrate-resistant acid phosphatase (TRACP)–positive multinucleated cells, TRACP activity in the medium, up-regulation of cathepsin K gene expression, and bone resorption. These inhibitions are associated with a down-regulation of RANK expression at both mRNA and cell surface protein levels and a decrease of NFATc1 stimulation and NF-κB nuclear translocation, whereas the gene expression of c-fms, CD14, and CD11a is up-regulated. Finally, resveratrol promotes dose-dependently the expression of osteoblast markers like osteocalcin and osteopontin in human bone marrow mesenchymal stem cells (hMSC-TERT) and stimulates their response to 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. Moreover, resveratrol up-regulates dose-dependently the expression of 1,25(OH)2D3 nuclear receptor. Taken together, these results suggest that resveratrol or its derivatives deserve attention as potential drugs for treating multiple myeloma.
Antiandrogen Bicalutamide Promotes Tumor Growth in a Novel Androgen-Dependent Prostate Cancer Xenograft Model Derived from a Bicalutamide-Treated Patient
Authors: Yoshida Toru , Kinoshita Hidefumi , Segawa Takehiko , Nakamura Eijiro , Inoue Takahiro , Shimizu Yousuke , Kamoto Toshiyuki , Ogawa Osamu
Published: November 2005

Androgen ablation therapies are effective in controlling prostate cancer. Although most cancers relapse and progress despite androgen ablation, some patients experience antiandrogen withdrawal syndrome, in which those treated with antiandrogen show clinical improvement when antiandrogen is discontinued. Although the androgen receptor (AR) is suggested to play an important role in prostate cancer progression even after the androgen ablation, limited tissue availability for molecular studies and small numbers of human prostate cancer cell lines have restricted prostate cancer research. Here, we describe KUCaP, a novel serially transplantable human prostate cancer xenograft model. We established KUCaP from liver metastatic tissue of a patient treated with antiandrogen bicalutamide. KUCaP expressed the AR with a point mutation at amino acid 741 (tryptophan to cysteine; W741C) in the ligand-binding domain. This mutation was also present in cancerous tissue used for generation of KUCaP. Although the growth of KUCaP in male mice was androgen dependent, bicalutamide aberrantly promoted the growth and prostate-specific antigen production of KUCaP. For the first time, we show the agonistic effect of bicalutamide to a xenograft with clinically induced AR mutation. This bicalutamide-responsive mutant AR will serve in the development of new therapies for androgen ablation–resistant prostate cancers.
Development of a Conditional In vivo Model to Evaluate the Efficacy of Small Molecule Inhibitors for the Treatment of Raf-Transformed Hematopoietic Cells
Authors: Konopleva Marina , Shi Yuexi , Steelman Linda S. , Shelton John G. , Munsell Mark , Marini Frank , McQueen Teresa , Contractor Rooha , McCubrey James A. , Andreeff Michael
Published: November 2005

Conditionally active forms of the Raf proteins (Raf-1, B-Raf, and A-Raf) were created by ligating NH2-terminal truncated activated forms (Δ) to the estrogen receptor (ER) hormone-binding domain resulting in estradiol-regulated constructs (ΔRaf:ER). These different Raf:ER oncoproteins were introduced into the murine FDC-P1 hematopoietic cell line, and cells that grew in response to the three ΔRaf:ER oncoproteins were isolated. The ability of FDC-P1, ΔRaf-1:ER, ΔA-Raf:ER, and ΔB-Raf:ER cells to form tumors in severe combined immunodeficient mice was compared. Mice injected with ΔRaf:ER cells were implanted with β-estradiol pellets to induce the ΔRaf:ER oncoprotein. Cytokine-dependent parental cell lines did not form tumors. Implantation of β-estradiol pellets into mice injected with ΔRaf:ER cells significantly accelerated tumor onset and tumor size. The recovered ΔRaf:ER cells displayed induction of extracellular signal-regulated kinase (ERK) in response to β-estradiol stimulation, indicating that they had retained conditional activation of ERK even when passed through a severe combined immunodeficient mouse. The ΔRaf:ER cells were very sensitive to induction of apoptosis by the mitogen-activated protein/ERK kinase (MEK) 1 inhibitor CI1040 whereas parental cells were much less affected, demonstrating that the MEK1 may be useful in eliminating Ras/Raf/MEK–transformed cells. Furthermore, the effects of in vivo administration of the MEK1 inhibitor were evaluated and this inhibitor was observed to suppress the tumorigenicity of the injected cells. This ΔRaf:ER system can serve as a preclinical model to evaluate the effects of signal transduction inhibitors which target the Raf and MEK proteins.
Myxoma Virus Is a Novel Oncolytic Virus with Significant Antitumor Activity against Experimental Human Gliomas
Authors: Lun Xueqing , Yang Wenqing , Alain Tommy , Shi Zhong-Qiao , Muzik Huong , Barrett John W. , McFadden Grant , Bell John , Hamilton Mark G. , Senger Donna L. , Forsyth Peter A.
Published: November 2005

Myxoma virus, a poxvirus previously considered rabbit specific, can replicate productively in a variety of human tumor cells in culture. The purpose of this study was to determine if there was efficacy or toxicities of this oncolytic virus against experimental models of human malignant gliomas in vitro, in vivo, and ex vivo in malignant glioma specimens. In vitro, the majority of glioma cell lines tested (7 of 8, 87.5%) were fully permissive for myxoma virus replication and killed by infection. In vivo, intracerebral (i.c.) myxoma virus inoculation was well tolerated and produced only minimal focal inflammatory changes at the site of viral inoculation. U87 and U251 orthotopic xenograft models were used to assess myxoma virus efficacy in vivo. A single intratumoral injection of myxoma virus dramatically prolonged median survival compared with treatment with UV-inactivated myxoma virus. Median survival was not reached in myxoma virus-treated groups versus 47.3 days (U87; P = 0.0002) and 50.7 days (U251; P = 0.0027) in UV-inactivated myxoma virus-treated groups. Most myxoma virus-treated animals (12 of 13, 92%) were alive and apparently “cured” when the experiment was finished (>130 days). Interestingly, we found a selective and long-lived myxoma virus infection in gliomas in vivo. This is the first demonstration of the oncolytic activity of myxoma virus in vivo. The nonpathogenic nature of myxoma virus outside of the rabbit host, its capacity to be genetically modified, its ability to produce a long-lived infection in human tumor cells, and the lack of preexisting antibodies in the human population suggest that myxoma virus may be an attractive oncolytic agent against human malignant glioma.
Genetic Ablation of Protein Tyrosine Phosphatase 1B Accelerates Lymphomagenesis of p53-Null Mice through the Regulation of B-Cell Development
Authors: Dubé Nadia , Bourdeau Annie , Heinonen Krista M. , Cheng Alan , Lee Loy Ailsa , Tremblay Michel L.
Published: November 2005

Protein tyrosine phosphatase 1B (PTP1B) is involved in multiple signaling pathways by down-regulating several tyrosine kinases. For example, gene-targeting studies in mice have established PTP1B as a critical physiologic regulator of metabolism by attenuating insulin signaling. PTP1B is an important target for the treatment of diabetes, because the PTP1B null mice are resistant to diet-induced diabetes and obesity. On the other hand, despite the potential for enhanced oncogenic signaling in the absence of PTP1B, PTP1B null mice do not develop spontaneous tumors. Because the majority of human cancers harbor mutations in p53, we generated p53/PTP1B double null mice to elucidate the role of PTP1B in tumorigenesis. We show that genetic ablation of PTP1B in p53 null mice decreases survival rate and increases susceptibility towards the development of B lymphomas. This suggested a role for PTP1B in lymphopoiesis, and we report that PTP1B null mice have an accumulation of B cells in bone marrow and lymph nodes, which contributed to the increased incidence of B lymphomas. The mean time of tumor development and tumor spectrum are unchanged in p53−/−PTP1B+/− mice. We conclude that PTP1B is an important determinant of the latency and type of tumors in a p53-deficient background through its role in the regulation of B-cell development.
Gene Expression Profiling IdentifiesBAX-δas a Novel Tumor Antigen in Acute Lymphoblastic Leukemia
Authors: Maia Sara , Haining W. Nicholas , Ansén Sascha , Xia Zhinan , Armstrong Scott A. , Seth Nilufer P. , Ghia Paolo , den Boer Monique L. , Pieters Rob , Sallan Stephen E. , Nadler Lee M. , Cardoso Angelo A.
Published: November 2005

The identification of new tumor-associated antigens (TAA) is critical for the development of effective immunotherapeutic strategies, particularly in diseases like B-cell acute lymphoblastic leukemia (B-ALL), where few target epitopes are known. To accelerate the identification of novel TAA in B-ALL, we used a combination of expression profiling and reverse immunology. We compared gene expression profiles of primary B-ALL cells with their normal counterparts, B-cell precursors. Genes differentially expressed by B-ALL cells included many previously identified as TAA in other malignancies. Within this set of overexpressed genes, we focused on those that may be functionally important to the cancer cell. The apoptosis-related molecule, BAX, was highly correlated with the ALL class distinction. Therefore, we evaluated BAX and its isoforms as potential TAA. Peptides from the isoform BAX-δ bound with high affinity to HLA-A*0201 and HLA-DR1. CD8+ CTLs specific for BAX-δ epitopes or their heteroclitic peptides could be expanded from normal donors. BAX-δ–specific T cells lysed peptide-pulsed targets and BAX-δ–expressing leukemia cells in a MHC-restricted fashion. Moreover, primary B-ALL cells were recognized by BAX-δ–specific CTL, indicating that this antigen is naturally processed and presented by tumor cells. This study suggests that (a) BAX-δ may serve as a widely expressed TAA in B-ALL and (b) gene expression profiling can be a generalizable tool to identify immunologic targets for cancer immunotherapy.
The Enhanced Tumor Selectivity of an Oncolytic Vaccinia Lacking the Host Range and Antiapoptosis Genes SPI-1 and SPI-2
Authors: Guo Z. Sheng , Naik Arpana , O'Malley Mark E. , Popovic Petar , Demarco Richard , Hu Yun , Yin Xiaoyu , Yang Shuting , Zeh Herbert J. , Moss Bernard , Lotze Michael T. , Bartlett David L.
Published: November 2005

The ability of cancer cells to evade apoptosis may permit survival of a recombinant vaccinia lacking antiapoptotic genes in cancer cells compared with normal cells. We have explored the deletion of two vaccinia virus host range/antiapoptosis genes, SPI-1 and SPI-2, for their effects on the viral replication and their ability to induce cell death in infected normal and transformed cells in vitro. Indeed, in three paired normal and transformed cell types, the SPI-1 and SPI-2 gene-deleted virus (vSP) preferentially replicates in transformed cells or p53-null cells when compared with their normal counterparts. This selectivity may be derived from the fact that vSP-infected normal cells died faster than infected cancer cells. A fraction of infected cells died with evidence of necrosis as shown by both flow cytometry and detection of high-mobility group B1 protein released from necrotic cells into the culture supernatant. When administered to animals, vSP retains full ability to replicate in tumor tissues, whereas replication in normal tissues is greatly diminished. In a model of viral pathogenesis, mice treated with vSP survived substantially longer when compared with mice treated with the wild-type virus. The mutant virus vSP displayed significant antitumoral effects in an MC38 s.c. tumor model in both nude (P < 0.001) and immunocompetent mice (P < 0.05). We conclude that this recombinant vaccinia vSP shows promise for oncolytic virus therapy. Given its enhanced tumor selectivity, improved safety profile, and substantial oncolytic effects following systemic delivery in murine models, it should also serve as a useful vector for tumor-directed gene therapy.
N-Benzyladriamycin-14-Valerate (AD198) Induces Apoptosis through Protein Kinase C-δ–Induced Phosphorylation of Phospholipid Scramblase 3
Authors: He Yongwen , Liu Jihua , Durrant David , Yang Hung-Sheng , Sweatman Trevor , Lothstein Leonard , Lee Ray M.
Published: November 2005

Phospholipid scramblase 3 (PLS3) is an enzyme that plays a critical role in mitochondrial morphology, functions, and apoptotic response. During apoptosis, activated protein kinase C-δ (PKC-δ) translocates to mitochondria and phosphorylates PLS3. Here, we utilize an extranuclear-targeted anthracycline N-benzyladriamycin-14-valerate (AD198), a PKC-δ activator, to investigate the mechanism of PLS3 phosphorylation by PKC-δ. Overexpression of PLS3 enhanced, whereas down-regulation of PLS3 by small interfering RNA decreased, the sensitivity of AD198-induced apoptosis. Overexpression of PKC-δ, but not the kinase-defective PKC-δ, and AD198 treatment enhanced threonine phosphorylation of PLS3. The phosphorylated threonine was mapped to Thr21 of PLS3. Mutation of Thr21 to alanine did not affect mitochondrial localization of PLS3 but abolished threonine phosphorylation by PKC-δ in vitro and AD198-induced PLS3 phosphorylation in vivo. Expression of PLS3(T21A) in cells could not enhance AD198-induced apoptosis compared with expression of the wild-type PLS3. Using benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone and cyclosporine A, we also showed that AD198-induced PLS3 phosphorylation occurs upstream of caspase activation and independent of mitochondrial permeability transition. These studies establish that AD198-activated PKC-δ induces phosphorylation of mitochondrial PLS3 at Thr21 and that PLS3 is a critical downstream effector of PKC-δ in AD198-induced apoptosis.
Tumor Cell Loaded Type-1 Polarized Dendritic Cells Induce Th1-Mediated Tumor Immunity
Authors: Hokey David A. , Larregina Adriana T. , Erdos Geza , Watkins Simon C. , Falo Louis D.
Published: November 2005

Dendritic cells are professional antigen-presenting cells capable of inducing and regulating innate and antigen-specific immune responses. Therapeutic cancer vaccines using ex vivo engineered or in vivo targeted dendritic cells are being evaluated in clinical trials. T-helper type-1 (Th1)–skewed immune responses are characterized by the preferential induction of antigen-specific IFN-γ–secreting CD4+ T cells and correlate with effector mechanisms important for tumor and viral immunity. Methods to “polarize” human monocyte-derived dendritic cells for the preferential induction of Th1-skewed immune responses have been developed, and polarized dendritic cells (DC1s) are being evaluated in preclinical and clinical studies. Here, we show that stimulation of bone marrow–derived murine dendritic cell populations with poly(I:C) and CpGs results in phenotypic maturation of dendritic cells and synergistic induction of durable, high-level IL-12p70 secretion characteristic of human type-1 polarized dendritic cells. Functionally, these dendritic cells induce antigen-specific Th1-type CD4+ T-cell activation in vitro and in vivo. Dendritic cell maturation and polarization are not inhibited by the presence of live B16 melanoma tumor cells, and tumor-loaded DC1s induce delayed-type hypersensitivity responses in vivo. DC1s loaded with B16 melanoma cells and injected into tumor-bearing mice induce Th1-skewed tumor-specific CD4+ T cells and a significant reduction in tumor growth. Tumor infiltrates in DC1-immunized animals are characterized by the presence of CD4+ T cells and activated macrophages. These results show a murine model of DC1 function and suggest an important role for CD4+ T cells and macrophages in DC1-induced antitumor immune responses. They have implications for the future development of DC1-based immunotherapies and strategies for clinical immune monitoring of their effectiveness.
DNA Damage Disrupts the p14ARF-B23(Nucleophosmin) Interaction and Triggers a Transient Subnuclear Redistribution of p14ARF
Authors: Lee Casey , Smith Brian A. , Bandyopadhyay Keya , Gjerset Ruth A.
Published: November 2005

The p14 alternate reading frame (ARF) tumor suppressor plays a central role in cancer by binding to mdm2 (Hdm2 in humans) and enhancing p53-mediated apoptosis following DNA damage and oncogene activation. It is unclear, however, how ARF initiates its involvement in the p53/mdm2 pathway, as p53 and mdm2 are located in the nucleoplasm, whereas ARF is largely nucleolar in tumor cells. We have used immunofluorescence and coimmunoprecipitation to examine how the subnuclear distribution and protein-protein interactions of ARF change immediately after DNA damage and over the time course of the DNA damage response in human tumor cells. We find that DNA damage disrupts the interaction of ARF with the nucleolar protein B23(nucleophosmin) and promotes a transient p53-independent translocation of ARF to the nucleoplasm, resulting in a masking of the ARF NH2 terminus that correlates with the appearance of ARF-Hdm2 complexes. The translocation also results in an unmasking of the ARF COOH terminus, suggesting that redistribution disrupts a nucleolar interaction of ARF involving this region. By 24 hours after irradiation, DNA repair has ceased and the pretreatment immunofluorescence patterns and complexes of ARF have been restored. Although the redistribution of ARF is independent of p53 and likely to be regulated by interactions other than Hdm2, ARF does not promote UV sensitization unless p53 is expressed. The results implicate the nucleolus and nucleolar interactions of the ARF, including potentially novel interactions involving its COOH terminus as sites for early DNA damage and stress-mediated cellular events.
A Novel Strategy for the Discovery of MHC Class II–Restricted Tumor Antigens: Identification of a Melanotransferrin Helper T-Cell Epitope
Authors: Röhn Till A. , Reitz Annette , Paschen Annette , Nguyen Xuan D. , Schadendorf Dirk , Vogt Anne B. , Kropshofer Harald
Published: November 2005

CD4+ helper T cells play a critical role in orchestrating host immune responses, including antitumor immunity. The limited availability of MHC class II–associated tumor antigens is still viewed as a major obstacle in the use of CD4+ T cells in cancer vaccines. Here, we describe a novel approach for the identification of MHC class II tumor-associated antigens (TAAs). By combining two-dimensional liquid chromatography and nanoelectrospray ionization tandem mass spectrometry, we developed a highly sensitive method for the detection of human leukocyte antigen (HLA)-DR–associated peptides of dendritic cells upon exposure to necrotic tumor cells. This approach led to the identification of a novel MHC class II–restricted TAA epitope derived from melanotransferrin. The epitope stimulated T cells derived from melanoma patients and healthy individuals and displayed promiscuity in HLA-DR restriction. Moreover, the same peptide was also presented by MHC class II–positive melanoma cells. This strategy may contribute to increase the number of tumor epitopes presented by MHC class II molecules and may support the development of more efficacious vaccines against cancer.