DOCSLIB.ORG

A Cost-Effectiveness Method for Detection of Abl Mutations in Patients Who Developed Imatinib Resistance

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Cost-Effectiveness Method for Detection of Abl Mutations in Patients Who Developed Imatinib Resistance WCRJ 2016; 3 (4): e799 A COST-EFFECTIVENESS METHOD FOR DETECTION OF ABL MUTATIONS IN PATIENTS WHO DEVELOPED IMATINIB RESISTANCE C. FIERRO1, T. MUTO1, M. TREMATERRA1, F. MORANO1, S. PUGLIESE2, M. DI PAOLO2, A. TROISI2, G. CRESCENTE3 1Hematology and Cellular Immunology Clinical Biochemystry A.O. dei Colli Monaldi, Naples Italy 2CETAC Research Centre, Caserta, Italy 3Italian Association of Pharmacogenomics and Molecular Diagnostics, Caserta, Italy Abstract – Background: Despite high response rate to specific tyrosine kinase inhibitors (TKI), resistances have been observed in patients in treatment with Imatinib because acquired mutations into BCR-ABL1 kinase domain (KD). This can prove a challenge and underlines the importance of utilizing a detection method that is easy, sensitive, reproducible and cost-effective. Materials and Methods: We developed a high specific and sensitive detection assay in order to quickly and easily identify T315I mutation in gene ABL patients by Peptide Nucleic Acid (PNA) directed PCR clamping. Results: The experimental design forecasts that both PNA and PCR primer mutant target sites overlap, thus leading to a direct competition towards complementary DNA (cDNA). 25 μM of ol- igo-PNA was enough to perfect matching PNA/cDNA duplex template in wild-type ABL sequence and PCR amplification is suppressed. Contemporary, in the mutant DNA (I315I) the PNA/cDNA du- plex hybridization fail and PCR can be performed. Conclusions: This detection method is easy, sensitive, reproducible and very cheap. Sequencing method could be restricted as confirmation method only in doubtful mutant samples. Finally, this platform should be ideal for small laboratory that processing few samples. KEYWORDS: PNA Clamping PCR, BCR-Abl, T315I, Imatinib, Sequencing, Chronic myeloid leukemia, Philadelphia chromosome. INTRODUCTION tyrosine kinase inhibitors2. Among different mu- tations identified the frequently observed T315I Chronic myeloid leukemia (CML) is character- is of particular concern since it is not effectively ized by the presence of the Philadelphia chro- targeted by the majority of TKIs so far available3. mosome (Ph+) resulting from a translocation be- The only drug showing activity against I315I tween chromosomes 9 and 221. Ph chromosome positive CML is ponatinib4. creates the BCR-ABL fusion gene coding for a Currently, the recommended method for BCR- constitutive active tyrosine kinase ABL protein. ABL mutation detection is the sequencing of the Despite high response rate to specific tyrosine KD of ABL gene from exon 4 to 10. This is time kinase inhibitor (TKI), primary and secondary consuming and it allows reaching a maximum resistance has been observed: upfront resistance sensitivity of 10-15% of mutant DNA in a large is defined as lack of initial response and acquired excess amount of wild-type (wt) DNA5. resistance is defined as loss of an established re- The latter point represents a limit, as fre- sponse. BCR-ABL kinase domain (KD) mutations quently mutated clones may be present at a lower represent a well-established cause of resistance to percentage6. Corresponding Author: Carla Fierro, BS, Ph.D; e-mail: [email protected] 1 A relative new technique such the “ultra deep TABLE 1. Most important methodics applied for screening sequencing” allows to reach a very high level of e identification of mutation in ABL. sensitivity but it is far from been routinely appli- Method Autor Analitic cable in world-wide laboratories7. sensibility The availability of a simple, sensitive and DNA sequencing Gorre et al12 10-20% quick assay, allowing a rapid detection of the Shah et al13 T315I mutation is therefore crucial, as the de- Nested PCR+ Brandford et al14 10% tection of this mutation represents an important Sequencing Hochhaus et al15 element in clinical decision for CML patients. D-HPLC Soverini et al16 17 Since far time are used different techniques Irving et al Deiniger et al18 capable of identifying individual polymorphisms. Ernst et al19 1-5 % Among these, some have received validation for Pyrosequencing Khorashad et al20 1-5% clinical/diagnostic purposes [Single-Strand Con- HRM Polàkovà et al21 1-5% formational Polymorphism (SSCP) and Restric- Double Gradient Sorel et al22 tion Fragment Length Polymorphism (RFLP)] Denaturing Electrophoresis and others are still in an experimental phase of MALDI-TOF Vivante et al23 validation [Denaturing High Performance Liq- Allele specific Roche-lestienne et al24 1% uid Chromatography (D-HPLC), High Resolution PCR Gruber et al25 Melt (HRM) GENE-chip, MALDI-TOF, PNA Pelzackermann et al26 27 Clamping PCR]. A good example of the compara- Scorpion Probe Khorashad et al 0.1 % PNA fluorescent- Kreuzer et al28 0.1% tive sensitivity and specificity of the method listed clamping-PCR is summarized on (Table 1), which reporting the screening of point mutations in the ABL gene in patients with CML. Among all the techniques for the discrimination of point mutations, the PNA plate amplification. Competitor PNA sequence Clamping PCR is of great interest as it is capable was designed to perfectly match wild-type (wt) of identifying minimal amounts of variant gene template sequence. Therefore, when a single base containing the mutation within a sample almost pair mismatch occurs (like in the case of T315I) exclusively “wild-type” (for example, identifica- PNA-template stability is strongly impaired and tion of small quantities of cancer cells bearing a DNA amplification favored. mutation within a biopsy where there is an excess In this report, we describe a new assay method of normal tissue)8. based on 3′-Peptide nucleic acid (PNA) and modi- Oligo-PNA is a potent DNA mimic in terms fied reverse primer able to clump the amplification of sequence specific with high hybridization of wild-type (wt) DNA. This detection method is affinity. The duplex PNA/DNA thermodynamic easy, sensitive, reproducible and cost-effective. is more stable than DNA/DNA or DNA/RNA This method should be perfect in terms of costs duplexes9, but PNA sequences cannot be ex- for small laboratory that processing few samples. tended by DNA polymerase10. As consequence, PNA/DNA duplex suppresses DNA PCR ampli- fication. Furthermore, PNA/DNA hybridization shows a greater single-base-pair mismatch dis- MATERIALS AND METHODS crimination than the corresponding DNA/DNA duplex. Positive controls Based on this premise and previous data, we developed a novel and sensitive detection assay Positive controls of the conventional PCR and in order to quickly and easily identify T315I sequencing are represented from the cellular mutation in CML patients by PNA directed PCR line K562 in which the gene of BCR is present clamping11. The experimental design forecasts in fusion BCR/ABL p210 (exon 14, exon 2 of that both PNA and PCR primer target sites over- Abl) with b3a2. The rearrangement controls for lap, thus leading to a direct competition towards the mutational screening of PNA Clamping PCR complementary DNA. When perfect matching are represent by a plasmid of 2710 bases (bp) occurs PNA/DNA template hybridization is fa- synthesized from the Gen company Script (The vored more than primer-DNA template duplex Biology Crow USA) in which it has been intro- and DNA amplification is suppressed. Converse- duced the sequence of 643 bp of the ABL gene ly, a single mismatch destabilizes the PNA/DNA comprised between exon 4and exon the 7, that it template duplex, favoring the hybridization be- contains the mutations that will come taken in tween template and primer thus allowing tem- consideration. 2 A COST-EFFECTIVENESS METHOD FOR DETECTION OF ABL MUTATIONS IN PATIENTS WHO DEVELOPED IMATINIB RESISTANCE Sampling 40U RNAasi inibitor (Life Technologies, Milan, Italy) and 200U of Reverse Transcriptase “Super White blood cells (WBC) from bone marrow Script III” (Life Technologies, Milan, Italy). The and peripheral blood are obtained for erithrolisys reaction was incubated for 60 minutes to 42°C using ipotonic solutions made up of ammonium and subsequently for 15 minutes to 75°C. chloride and potassium bicarbonate. The pellet cellular it comes washed in PBS (NaCl 137 mM, KCl 2.7 mM, Na2HPO4.7H 2O, 8.1 mM, KH2PO4 Selective amplification of transcribed 1.9 mM) and quoted in 2 tubes for the extraction fusion gene BCR/ABL of nucleic acids. For the extraction of the DNA the pellet it comes frozen to -20°C, while for the For the mutational screening uses a amplicon extraction of the RNA, the share it comes mixture obtained from a conventional PCR that uses a in phenol/guanidinic acid solution (TRIZOL Invi- primer that it recognizes the gene in the region of trogen, Milan, Italy) and frozen to -20°C. the Major-Breakpoint Cluster Region (M-BCR), post on exon the 12 of BCR and a designed other on exon 7 of ABL. The Forward BCR exon 12 Isolation of RNA primer sequence is 5’-TCC GCT GAC CAT CAA TAA GG-3’ and ABL Reverse 5’-CCA GAC GTC For the extraction of the RNA total, approximate- GGA CTT GAT GG-3. ly 10 million of WBC was combined to 1 mL of This PCR product is used as template for the TRIZOL (Invitrogen, Milan, Italy), added 0.2 mL next PCR-screening for one requirement: of Clorophorme. After centrifugation to 12.000 g 1. To increase the sensibility of amplification for 15 minute to 4°C was evident two phases: one 2. To select the allele BCR-ABL rearrangement organic (down) and one watery (upper). The water in the neoplastic cells excluding the wild-type phase overhanging comes captured and added to ABL (that it major increases the specificity of one equal amount of isopropanol. The nucleic the analysis). acid falls after centrifuge under shape of knows The amplification was performed with 1 μl them. Eventual polluting agents are efficiently of cDNA in a mixture of 25 mL of 10 x Buf- washing by successive 75%ethanol step. The pel- fer (Roche, Milan, Italy) in which is contained let of RNA is resolved in 30 mL of RNAasi-free 1.5mm MgCl2, 200 nM of both primers, 200 nM water.
Load more

Recommended publications
  • Gene Symbol Gene Description ACVR1B Activin a Receptor, Type IB
    Table S1. Kinase clones included in human kinase cDNA library for yeast two-hybrid screening Gene Symbol Gene Description ACVR1B activin A receptor, type IB ADCK2 aarF domain containing kinase 2 ADCK4 aarF domain containing kinase 4 AGK multiple substrate lipid kinase;MULK AK1 adenylate kinase 1 AK3 adenylate kinase 3 like 1 AK3L1 adenylate kinase 3 ALDH18A1 aldehyde dehydrogenase 18 family, member A1;ALDH18A1 ALK anaplastic lymphoma kinase (Ki-1) ALPK1 alpha-kinase 1 ALPK2 alpha-kinase 2 AMHR2 anti-Mullerian hormone receptor, type II ARAF v-raf murine sarcoma 3611 viral oncogene homolog 1 ARSG arylsulfatase G;ARSG AURKB aurora kinase B AURKC aurora kinase C BCKDK branched chain alpha-ketoacid dehydrogenase kinase BMPR1A bone morphogenetic protein receptor, type IA BMPR2 bone morphogenetic protein receptor, type II (serine/threonine kinase) BRAF v-raf murine sarcoma viral oncogene homolog B1 BRD3 bromodomain containing 3 BRD4 bromodomain containing 4 BTK Bruton agammaglobulinemia tyrosine kinase BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) BUB1B BUB1 budding uninhibited by benzimidazoles 1 homolog beta (yeast) C9orf98 chromosome 9 open reading frame 98;C9orf98 CABC1 chaperone, ABC1 activity of bc1 complex like (S. pombe) CALM1 calmodulin 1 (phosphorylase kinase, delta) CALM2 calmodulin 2 (phosphorylase kinase, delta) CALM3 calmodulin 3 (phosphorylase kinase, delta) CAMK1 calcium/calmodulin-dependent protein kinase I CAMK2A calcium/calmodulin-dependent protein kinase (CaM kinase) II alpha CAMK2B calcium/calmodulin-dependent
    [Show full text]
  • Cytokine Signaling Through the Novel Tyrosine Kinase RAFTK in Kaposi's
    Cytokine Signaling Through the Novel Tyrosine Kinase RAFTK in Kaposi’s Sarcoma Cells Zhong-Ying Liu,* Ramesh K. Ganju,*Jian-Feng Wang,* Mel A. Ona,* William C. Hatch,* Tong Zheng,‡ Shalom Avraham,* Parkash Gill,‡ and Jerome E. Groopman* *Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; and ‡Division of Hematology/Oncology, Norris Cancer Center, University of Southern California, Los Angeles, California 90033 Abstract believed to be from the lymphatic endothelium (1–2). Etiolog- ical factors implicated in KS include the recently discovered A number of cytokines, including basic fibroblast growth human herpesvirus 8 (HHV-8)/Kaposi’s sarcoma herpesvirus factor (bFGF), vascular endothelial growth factor (VEGF), (KSHV) and TAT, the soluble transcriptional activator of oncostatin M (OSM), IL-6, and tumor necrosis factor alpha HIV (3–7). Considerable data indicate a role for endogenous (TNF-a), have been postulated to have a role in the patho- and exogenous cytokines in the pathogenesis of KS (8–16). genesis of Kaposi’s sarcoma (KS). The proliferative effects Growth factors such as basic fibroblast growth factor (bFGF) of bFGF and OSM may be via their reported activation of and vascular endothelial growth factor (VEGF), which are the c-Jun NH2-terminal kinase (JNK) signaling pathway in known to stimulate the mitogenesis of certain types of endo- KS cells. We now report that KS cells express a recently thelium, as well as Oncostatin M (OSM), IL-6, and tumor ne- identified focal adhesion kinase termed RAFTK which ap- crosis factor alpha (TNF-a) which are elaborated during in- pears in other cell systems to coordinate surface signals be- flammatory conditions, have been implicated in promoting KS tween cytokine and integrin receptors and the cytoskeleton cell growth (17–25).
    [Show full text]
  • Itk Tyrosine Kinase Substrate Docking Is Mediated by a Nonclassical SH2 Domain Surface of PLC␥1
    Itk tyrosine kinase substrate docking is mediated by a nonclassical SH2 domain surface of PLC␥1 Lie Min, Raji E. Joseph, D. Bruce Fulton, and Amy H. Andreotti1 Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011 Edited by Susan S. Taylor, University of California at San Diego, La Jolla, CA, and approved October 20, 2009 (received for review October 1, 2009) Interleukin-2 tyrosine kinase (Itk) is a Tec family tyrosine kinase that We have previously shown that the PLC␥1 SH2C domain mediates signaling processes after T cell receptor engagement. Acti- (spanning residues 659–756 within full-length PLC␥1) binds di- vation of Itk requires recruitment to the membrane via its pleckstrin rectly to the Itk kinase domain and is required for efficient homology domain, phosphorylation of Itk by the Src kinase, Lck, and phosphorylation of Y783 by Itk (26). Fragments of PLC␥1 that binding of Itk to the SLP-76/LAT adapter complex. After activation, Itk contain Y783 but not the SH2C domain are not efficiently phos- phosphorylates and activates phospholipase C-␥1 (PLC-␥1), leading to phorylated by Itk. Moreover, phosphorylation of PLC␥1 substrate production of two second messengers, DAG and IP3. We have previ- fragments that contain both SH2C and Y783 (spanning 659–789, ously shown that phosphorylation of PLC-␥1 by Itk requires a direct, hereafter referred to as PLC␥1 SH2C-linker) can be inhibited by phosphotyrosine-independent interaction between the Src homol- titration with isolated PLC␥1 SH2C domain (26). The excess, free ogy 2 (SH2) domain of PLC-␥1 and the kinase domain of Itk.
    [Show full text]
  • Supplementary Table 1. in Vitro Side Effect Profiling Study for LDN/OSU-0212320. Neurotransmitter Related Steroids
    Supplementary Table 1. In vitro side effect profiling study for LDN/OSU-0212320. Percent Inhibition Receptor 10 µM Neurotransmitter Related Adenosine, Non-selective 7.29% Adrenergic, Alpha 1, Non-selective 24.98% Adrenergic, Alpha 2, Non-selective 27.18% Adrenergic, Beta, Non-selective -20.94% Dopamine Transporter 8.69% Dopamine, D1 (h) 8.48% Dopamine, D2s (h) 4.06% GABA A, Agonist Site -16.15% GABA A, BDZ, alpha 1 site 12.73% GABA-B 13.60% Glutamate, AMPA Site (Ionotropic) 12.06% Glutamate, Kainate Site (Ionotropic) -1.03% Glutamate, NMDA Agonist Site (Ionotropic) 0.12% Glutamate, NMDA, Glycine (Stry-insens Site) 9.84% (Ionotropic) Glycine, Strychnine-sensitive 0.99% Histamine, H1 -5.54% Histamine, H2 16.54% Histamine, H3 4.80% Melatonin, Non-selective -5.54% Muscarinic, M1 (hr) -1.88% Muscarinic, M2 (h) 0.82% Muscarinic, Non-selective, Central 29.04% Muscarinic, Non-selective, Peripheral 0.29% Nicotinic, Neuronal (-BnTx insensitive) 7.85% Norepinephrine Transporter 2.87% Opioid, Non-selective -0.09% Opioid, Orphanin, ORL1 (h) 11.55% Serotonin Transporter -3.02% Serotonin, Non-selective 26.33% Sigma, Non-Selective 10.19% Steroids Estrogen 11.16% 1 Percent Inhibition Receptor 10 µM Testosterone (cytosolic) (h) 12.50% Ion Channels Calcium Channel, Type L (Dihydropyridine Site) 43.18% Calcium Channel, Type N 4.15% Potassium Channel, ATP-Sensitive -4.05% Potassium Channel, Ca2+ Act., VI 17.80% Potassium Channel, I(Kr) (hERG) (h) -6.44% Sodium, Site 2 -0.39% Second Messengers Nitric Oxide, NOS (Neuronal-Binding) -17.09% Prostaglandins Leukotriene,
    [Show full text]
  • FER Tyrosine Kinase (FER) Overexpression Mediates Resistance to Quinacrine Through EGF-Dependent Activation of NF-Κb
    FER tyrosine kinase (FER) overexpression mediates resistance to quinacrine through EGF-dependent activation of NF-κB Canhui Guo and George R. Stark1 Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195 Contributed by George R. Stark, April 5, 2011 (sent for review January 17, 2011) Quinacrine, a drug with antimalarial and anticancer activities that that overexpression of FER activates NF-κB, thus conferring inhibits NF-κB and activates p53, has progressed into phase II clin- resistance to the NF-κB inhibitor quinacrine. ical trials in cancer. To further elucidate its mechanism of action and identify pathways of drug resistance, we used an unbiased Results method for validation-based insertional mutagenesis to isolate Identification of FER in a Quinacrine-Resistant Clone. Eighteen dif- a quinacrine-resistant cell line in which an inserted CMV promoter ferent pools of human colon cancer RKO cells were infected with drives overexpression of the FER tyrosine kinase (FER). Overex- three different VBIM viruses (6) using a total of 1 million cells. pression of FER from a cDNA confers quinacrine resistance to sev- After propagation, each pool was replated and treated with 10 eral different types of cancer cell lines. We show that quinacrine μM quinacrine for 48 h. Twenty quinacrine-resistant colonies kills cancer cells primarily by inhibiting the activation of NF-κB and were observed 2 wk later in seven of the pools. The VBIM vectors that increased activation of NF-κB through FER overexpression contain LoxP sites, allowing excision of the promoter in candidate mediates resistance. EGF activates NF-κB and stimulates phosphor- mutant clones.
    [Show full text]
  • Protein Tyrosine Kinases: Their Roles and Their Targeting in Leukemia
    cancers Review Protein Tyrosine Kinases: Their Roles and Their Targeting in Leukemia Kalpana K. Bhanumathy 1,*, Amrutha Balagopal 1, Frederick S. Vizeacoumar 2 , Franco J. Vizeacoumar 1,3, Andrew Freywald 2 and Vincenzo Giambra 4,* 1 Division of Oncology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada; [email protected] (A.B.); [email protected] (F.J.V.) 2 Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada; [email protected] (F.S.V.); [email protected] (A.F.) 3 Cancer Research Department, Saskatchewan Cancer Agency, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada 4 Institute for Stem Cell Biology, Regenerative Medicine and Innovative Therapies (ISBReMIT), Fondazione IRCCS Casa Sollievo della Sofferenza, 71013 San Giovanni Rotondo, FG, Italy * Correspondence: [email protected] (K.K.B.); [email protected] (V.G.); Tel.: +1-(306)-716-7456 (K.K.B.); +39-0882-416574 (V.G.) Simple Summary: Protein phosphorylation is a key regulatory mechanism that controls a wide variety of cellular responses. This process is catalysed by the members of the protein kinase su- perfamily that are classified into two main families based on their ability to phosphorylate either tyrosine or serine and threonine residues in their substrates. Massive research efforts have been invested in dissecting the functions of tyrosine kinases, revealing their importance in the initiation and progression of human malignancies. Based on these investigations, numerous tyrosine kinase inhibitors have been included in clinical protocols and proved to be effective in targeted therapies for various haematological malignancies.
    [Show full text]
  • P94fer Facilitates Cellular Recovery of Gamma Irradiated Pre-T Cells
    Oncogene (1997) 14, 2871 ± 2880 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 p94fer facilitates cellular recovery of gamma irradiated pre-T cells S Halachmy, O Bern, L Schreiber, M Carmel, Y Sharabi, J Shoham and U Nir Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel p94fer is a ubiquitous, nuclear and cytoplasmic tyrosine These three kinases encompass a group of the known, kinase, whose accumulation has been demonstrated in all SH2 containing mammalian nuclear tyrosine kinases mammalian cell lines analysed. In the present work, the (Yates et al., 1995; Wilks and Kurban, 1988; Van Etten p94fer expression pro®le was determined in cell lines et al., 1989; Hao et al., 1989, 1991) which are not src which were not tested before. While being present in related. While p94fer and c-Fes share similar structures, several hematopoietic and non hematopoietic cell lines containing a SH2 domain which is ¯anked by an including thymic stromal cells, the p94fer kinase could not extended N-terminal tail (Wilks and Kurban, 1988; be detected in pre-T and T cell lines. p94fer was also Hao et al., 1989), the c-Abl kinase contains both SH2 absent in pre-B line, but accumulated in these cells upon and SH3 domains and an extended C-terminal portion their induced development to antibody producing cells. (Ren et al., 1994). Several functional motifs have been This is in agreement with the absence of p94fer in primary de®ned in the Abl c-terminus, including a nuclear thymic and splenic T lymphocytes and its induced localization signal (NLS) (Van Etten et al., 1989), accumulation in stimulated B cells.
    [Show full text]
  • Mammary Gland Specific Expression of Brk/PTK6 Promotes Delayed
    Lofgren et al. Breast Cancer Research 2011, 13:R89 http://breast-cancer-research.com/content/13/5/R89 RESEARCHARTICLE Open Access Mammary gland specific expression of Brk/PTK6 promotes delayed involution and tumor formation associated with activation of p38 MAPK Kristopher A Lofgren1,2,3, Julie H Ostrander1,3, Daniel Housa1,3,4, Gregory K Hubbard1,3, Alessia Locatelli1,3, Robin L Bliss3, Kathryn L Schwertfeger2,3,5 and Carol A Lange1,2,3,6* Abstract Introduction: Protein tyrosine kinases (PTKs) are frequently overexpressed and/or activated in human malignancies, and regulate cancer cell proliferation, cellular survival, and migration. As such, they have become promising molecular targets for new therapies. The non-receptor PTK termed breast tumor kinase (Brk/PTK6) is overexpressed in approximately 86% of human breast tumors. The role of Brk in breast pathology is unclear. Methods: We expressed a WAP-driven Brk/PTK6 transgene in FVB/n mice, and analyzed mammary glands from wild-type (wt) and transgenic mice after forced weaning. Western blotting and immunohistochemistry (IHC) studies were conducted to visualize markers of mammary gland involution, cell proliferation and apoptosis, as well as Brk, STAT3, and activated p38 mitogen-activated protein kinase (MAPK) in mammary tissues and tumors from WAP-Brk mice. Human (HMEC) or mouse (HC11) mammary epithelial cells were stably or transiently transfected with Brk cDNA to assay p38 MAPK signaling and cell survival in suspension or in response to chemotherapeutic agents. Results: Brk-transgenic dams exhibited delayed mammary gland involution and aged mice developed infrequent tumors with reduced latency relative to wt mice.
    [Show full text]
  • Rare Genetic Variants Potentially Involved in Ovarian Hyperstimulation Syndrome
    Journal of Assisted Reproduction and Genetics (2019) 36:491–497 https://doi.org/10.1007/s10815-018-1372-5 GENETICS Rare genetic variants potentially involved in ovarian hyperstimulation syndrome Katrien Stouffs1 & Sari Daelemans2 & Samuel Santos-Ribeiro2 & Sara Seneca1 & Alexander Gheldof1 & Ali Sami Gürbüz3 & Michel De Vos2 & Herman Tournaye2 & Christophe Blockeel2 Received: 25 May 2018 /Accepted: 14 November 2018 /Published online: 27 November 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract Purpose We aim to investigate whether there is a genetic predisposition in women who developed ovarian hyperstimulation syndrome (OHSS) after GnRH antagonist protocol with GnRH agonist trigger and freeze-all approach. Methods Four patients with OHSS after GnRH agonist trigger and freeze-all approach were gathered from the worldwide patient population. These patients were analyzed through Whole Exome Sequencing. In this study known causes of OHSS were investigated and new causes present in at least two individuals were searched for. Results In the first part of the study, we evaluated the presence of mutations in genes already known to be involved in OHSS. In PGR and TP53, heterozygous alterations were detected. PGR is predicted to be involved in progesterone resistance with a recessive inheritance pattern and is, therefore, not considered as being causal. The consequences of the variant detected in TP53 currently remain unknown. In part 2 of the study, we assessed the clinical significance of variants in genes previously not linked to OHSS. We especially focused on genes with variants present in ≥ 2 patients. Two patients have variants in the FLT4 gene. Mutations in this gene are linked to hereditary lymphedema, but no link to OHSS has been described.
    [Show full text]
  • Degradation of HER2/Neu by ANT2 Shrna Suppresses Migration and Invasiveness of Breast Cancer Cells Ji-Young Jang, Yoon-Kyung Jeon, Chul-Woo Kim*
    Jang et al. BMC Cancer 2010, 10:391 http://www.biomedcentral.com/1471-2407/10/391 RESEARCH ARTICLE Open Access Degradation of HER2/neu by ANT2 shRNA suppresses migration and invasiveness of breast cancer cells Ji-Young Jang, Yoon-Kyung Jeon, Chul-Woo Kim* Abstract Background: In breast cancer, the HER2/neu oncoprotein, which belongs to the epidermal growth factor receptor family, may trigger activation of the phosphoinositide-3 kinase (PI3K)/Akt pathway, which controls cell proliferation, survival, migration, and invasion. In this study, we examined the question of whether or not adenine nucleotide translocase 2 (ANT2) short hairpin RNA (shRNA)-mediated down-regulation of HER2/neu and inhibitory effects on the PI3K/Akt signaling pathway suppressed migration and invasiveness of breast cancer cells. Methods: We utilized an ANT2 vector-based RNA interference approach to inhibition of ANT2 expression, and the HER2/neu-overexpressing human breast cancer cell line, SK-BR3, was used throughout the study. Results: In this study, ANT2 shRNA decreased HER2/neu protein levels by promoting degradation of HER2/neu protein through dissociation from heat shock protein 90 (HSP90). As a result, ANT2 shRNA induced inhibitory effects on the PI3K/Akt signaling pathway. Inhibition of PI3K/Akt signaling by ANT2 shRNA caused down-regulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF) expression, decreased matrix metalloproteinase 2 (MMP2) and MMP9 activity, and suppressed migration and invasion of breast cancer cells. Conclusions: These results indicate that knock-down of ANT2 by shRNA down-regulates HER2/neu through suppression of HSP90’s function and inhibits the PI3K/Akt signaling pathway, resulting ultimately in suppressed migration and invasion of breast cancer cells.
    [Show full text]
  • Downloaded from Bioscientifica.Com at 09/23/2021 04:27:16PM Via Free Access Bernhard Et Al.: HER-2/Neu Vaccines
    Endocrine-Related Cancer (2002) 9 33–44 Vaccination against the HER-2/neu oncogenic protein H Bernhard1, L Salazar 2, K Schiffman2, A Smorlesi 2,3, B Schmidt1, K L Knutson2 andMLDisis2 1Technical University of Munich, Klinikum rechts der Isar, Department of Hematology and Oncology, Ismaningerstrasse 22, D-81664, Munich, Germany 2Division of Oncology, University of Washington, Seattle, Washington 98195–6527, USA 3Dipartimento Richerche INRCA, via Birarelli, 8, 60100 Ancona, Italy (Requests for offprints should be addressed to M L Disis, Box 356527, Oncology, University of Washington, Seattle, Washington 98195–6527, USA; Email: [email protected]) Abstract The HER-2/neu oncogenic protein is a well-defined tumor antigen. HER-2/neu is a shared antigen among multiple tumor types. Patients with HER-2/neu protein-overexpressing breast, ovarian, non- small cell lung, colon, and prostate cancers have been shown to have a pre-existent immune response to HER-2/neu. No matter what the tumor type, endogenous immunity to HER-2/neu detected in cancer patients demonstrates two predominant characteristics. First, HER-2/neu-specific immune responses are found in only a minority of patients whose tumors overexpress HER-2/neu. Secondly, immunity, if detectable, is of low magnitude. These observations have led to the develop- ment of vaccine strategies designed to boost HER-2/neu immunity in a majority of patients. HER-2/ neu is a non-mutated self-protein, therefore vaccines must be developed based on immunologic principles focused on circumventing tolerance, a primary mechanism of tumor immune escape. HER-2/neu-specific vaccines have been tested in human clinical trials.
    [Show full text]
  • Downloads.Action)
    bioRxiv preprint doi: https://doi.org/10.1101/259192; this version posted February 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. INKA, an integrative data analysis pipeline for phosphoproteomic inference of active phosphokinases Thang V. Pham1,2, Robin Beekhof1,2, Carolien van Alphen1,2, Jaco C. Knol1, Alex A. Henneman1, Frank Rolfs1, Mariette Labots1, Evan Henneberry1, Tessa Y.S. Le Large1, Richard R. de Haas1, Sander R. Piersma1, Henk M.W. Verheul1, Connie R. Jimenez1 * 1 OncoProteomics Laboratory, Department of Medical Oncology, Cancer Center Amsterdam, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands 2 These authors contributed equally to this work * Correspondence should be addressed to C.R.J. ([email protected]) Running Title: Integrative tool ranks active kinases Character Count: 50,356 1 bioRxiv preprint doi: https://doi.org/10.1101/259192; this version posted February 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Abstract 2 3 Identifying (hyper)active kinases in cancer patient tumors is crucial to enable individualized 4 treatment with specific inhibitors. Conceptually, kinase activity can be gleaned from global 5 protein phosphorylation profiles obtained with mass spectrometry-based phosphoproteomics. A 6 major challenge is to relate such profiles to specific kinases to identify (hyper)active kinases that 7 may fuel growth/progression of individual tumors. Approaches have hitherto focused on 8 phosphorylation of either kinases or their substrates.
    [Show full text]