DOCSLIB.ORG

An Epigenetically Derived Monoclonal Origin for Recurrent Respiratory Papillomatosis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
An Epigenetically Derived Monoclonal Origin for Recurrent Respiratory Papillomatosis ORIGINAL ARTICLE An Epigenetically Derived Monoclonal Origin for Recurrent Respiratory Papillomatosis Josena Kunjoonju Stephen, MD; Lori E. Vaught, MD; Kang Mei Chen, MD; Veena Shah, MD; Vanessa G. Schweitzer, MD; Glendon Gardner, MD; Michael S. Benninger, MD; Maria J. Worsham, PhD Objective: To investigate the contribution of pro- cer genes in the multigene panel had altered DNA meth- moter methylation-mediated epigenetic events in recur- ylation in at least 1 laryngeal papilloma biopsy speci- rent respiratory papillomatosis tumorigenesis. men. Identical abnormally methylated genes were found in 5 of 15 recurrent cases, of which the CDKN2B gene Design: Archival tissue DNA, extracted from microdis- was hypermethylated in all 5 cases. Dissimilar epige- sected papilloma lesions, was interrogated for methyl- netic events were noted in the remaining cases. ation status by means of the novel, multigene methylation- specific multiplex ligation-dependent probe amplification Conclusions: A clonal origin was derived for 5 of 15 assay. recurrent respiratory papillomatosis biopsy specimens based on identical epigenetic events. The high fre- Subjects: Fifteen subjects with recurrent respiratory pap- quency of epigenetic events, characterized by consis- illomatosis, 3 females and 12 males, all with adult onset tent promoter hypermethylation of multiple tumor of illness (age range, 23-73 years) except for 1 female pa- suppressor genes, points to the use of gene silencing tient with juvenile onset (1 year old). mechanisms in the pathogenesis of recurrent respira- tory papillomatosis. Results: Promoter hypermethylation was recorded in 14 of 15 cases, and 19 of 22 unique methylation-prone can- Arch Otolaryngol Head Neck Surg. 2007;133(7):684-692 ECURRENT RESPIRATORY small percentage of RRP cases progress to (laryngeal) papillomatosis malignancy.9 (RRP), an extremely rare Laryngeal papillomas usually run a be- condition, is characterized nign but recurrent course. Spontaneous by benign neoplasms transformation of RRP to squamous cell car- Rwithin the respiratory tract and can be cinoma is not easily characterized by a his- potentially life threatening because of tologic progression through dysplasia over airway obstruction.1 Recurrent respiratory time, making these lesions difficult to di- papillomatosis presents primarily as tiny or agnose histologically and clinically early in larger warts on the vocal chords. Preva- the course of the transformation of the lence of RRP worldwide is approximately disease. 100 000, with 2300 new cases in the United Clonality, the property that the cells States each year.2-4 Juvenile-onset disease oc- within a tumor are derived from a single curs in patients from younger than 1 year parent cell, is often indicated by unifor- to 8 years old; shows no sex difference5,6; mity or relative uniformity of genetic ab- has a rapid but often unpredictable pattern errations contained within many or all cells of recurrence5; tends to be a long-term, of- of the tumor. Such aberrations are as- ten lifelong disease; and exhibits a con- sumed to confer or reflect biological dis- Author Affiliations: Research tinuum of severity and aggressiveness. The tinctions relevant to tumor behavior, and Division, Department of adult form of RRP has a variable age at on- thus to be relevant to tumor initiation and Otolaryngology–Head and Neck set (peak, approximately 20-30 years),6,7 clonal expansion.10-13 Surgery (Drs Stephen, Vaught, 6 Chen, Schweitzer, Gardner, with a higher incidence in males. The se- Epigenetics is the regulation of changes Benninger, and Worsham), and verity, aggressiveness, and recurrence of the in gene expression by mechanisms that do Department of Pathology adult form tend to be less than in the juve- not involve changes in DNA sequence. Es- (Dr Shah), Henry Ford nile form.6 Human papillomavirus types 6 tablishment and maintenance of epige- Hospital, Detroit, Michigan. and 11 account for 80% to 90% of RRP.8 A netic control (gene silencing) has several (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 133 (NO. 7), JULY 2007 WWW.ARCHOTO.COM 684 ©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 MS-MLPA: Hhal digest Amplification of methylated target Antisense primer Antisense primer Labeled sense primer Labeled sense primer Ligation PCR Stuffer sequence Stuffer sequence with variable length Methylated with variable length Target DNA target CG CH Undigested by Methylated → no digestion → ligation 3 Antisense HhaI, binds to → Antisense primer probe PCR primer Labeled sense primer Labeled Hhal sense primer No PCR digestion Unmethylated target Target DNA CG Unmethylated → cut by Hhal → no ligation Digested by HhaI → does not bind to probe → no PCR Figure 1. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA).17 Probes designed to recognize HhaI sites within unmethylated regions will not generate a signal because these sequences have become cut by HhaI and cannot bind to the probe. Conversely, an MLPA probe will bind to an intact methylated site, spared by HhaI, and generate an amplification signal, producing 15 separate peaks (Figure 2) in a normal control DNA sample. Aberrant methylation is identified as the appearance of a signal peak that is otherwise absent in normal DNA samples (Figures 2, 3, and 4). CH3 indicates methyl group; PCR, polymerase chain reaction. aspects, which include promoter region hypermethyl- lowed to cool to 60°C. Next, 6 µL of 20-mg/mL proteinase K ation, methyl-binding proteins, DNA methyltransfer- was added, mixed, overlaid with 3 drops of mineral oil, and ases, histone deacetylases, and chromatin state. Aber- spun for 5 seconds at 13 000g. This was followed by a 4- to 16- rant methylation of CpG islands is a hallmark of human hour (overnight) incubation at 60°C. The tube was heated for cancers and is found early during carcinogenesis.14 Genes 10 minutes at 90°C to denature the proteinase K and to dis- rupt nucleic acid formaldehyde adducts. On removal of the oil, in cellular pathways that are inactivated by promoter re- the tube was centrifuged for 15 minutes (at 13 000g) at room gion hypermethylation include MGMT (DNA repair), temperature. Next, 250 µL of the supernatant was transferred INK4a INK4b p16 , p15 (cell cycle), DAPK (apoptosis), and to a clean 1.5-mL tube. On addition of 10 µL of 5M sodium GSTP1 (detoxification).15 chloride and 1000 mL of ethanol to the 250-µL supernatant, We investigated alterations in DNA methylation in bi- the tube was incubated at −20°C for least 60 minutes. This was opsy specimens of recurrences from patients with RRP followed by centrifugation for 15 minutes at 13 000g at −4°C. to assess the contribution of promoter methylation- On removal of the supernatant, an additional centrifugation step mediated epigenetic events in RRP tumorigenesis. for 10 seconds ensured removal of the last traces of the super- Aberrant promoter methylation of 22 methylation- natant. Finally, the pellet was air dried and dissolved in 100 prone tumor suppressor genes was evaluated by means µL of double-distilled water. of a high-throughput multigene probe panel (41 gene probes, 35 unique genes, including control probes) in MS-MLPA ASSAY 15 RRP cases by using the methylation-specific multi- The MS-MLPA assay allows for the relative quantification of ap- plex ligation-dependent probe amplification (MS-MLPA) 16,17 proximately 41 different DNA sequences in a single reaction re- assay. quiring only 20 ng of human DNA. The standard use of the tech- nique to observe quantitative changes in copy number has been METHODS outlined in other studies.18-21 Adaptation of the MLPA to detect aberrant methylation (MS-MLPA) has been detailed elsewhere.16,17 The probe design is similar to that of ordinary MLPA probes. RRP COHORT For 26 of 41 probes, the recognition sequence detected by the MLPA probe is contained within a restriction site for the methyl- The RRP cohort comprised 15 subjects, 3 females and 12 males, sensitive enzyme HhaI(Figure 1). all with adult onset of respiratory papillomatosis (age range, The41-gene-probepanel(Table 1)interrogates35uniquegenes 23-73 years) except for 1 female patient with juvenile onset (1 implicated in cancer, including head and neck squamous cell car- year old). The number of biopsy specimens from patients with cinoma, for losses and gains in a separate reaction in the absence recurrences ranged from 1 to 6. Archival tissue DNA, ex- of the methyl-sensitive enzyme HhaI. Because there are 2 probes tracted from microdissected papilloma lesions, was interro- eachforVHL,CDKN2A,BRCA1,andBRCA2,and3probesforMLH1, gated for methylation status by means of the MS-MLPA assay. a normal control DNA sample will generate 41 individual peaks in the absence of HhaI(Figure 2). A concurrently run reaction DNA EXTRACTION with the 41-gene-probe set in the presence of HhaI is designed to detect aberrant promoter hypermethylation by taking advantage As a first step, 300 µL of P-buffer (50mM Tris hydrochloride, of an HhaI site in the promoter region of 22 of the 35 unique genes pH 8.5; 100mM sodium chloride; 1mM EDTA; 0.5% Triton (note that 1 of the 2 BRCA1 probes is designed to recognize a re- X100; 20mM dithiothreitol) was added to tubes containing whole gion outside the HhaI recognition site; Table 1). Fifteen of the 41 5-µm tissue sections or microdissected tissue. The tube was gene probes are designed outside an HhaI site and serve as undi- heated for 15 to 20 minutes at 90°C in a water bath and al- gested controls (Figure 2). On digestion of the sample DNA with (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 133 (NO. 7), JULY 2007 WWW.ARCHOTO.COM 685 ©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 and 1 minute at 72°C; and 30 cycles of 30 seconds at 95°C, 30 Table 1.
Load more

Recommended publications
  • Synergistic Tumor Suppression by Combined Inhibition of Telomerase
    Synergistic tumor suppression by combined inhibition PNAS PLUS of telomerase and CDKN1A Romi Guptaa, Yuying Donga, Peter D. Solomona, Hiromi I. Wetterstenb, Christopher J. Chengc,d, JIn-Na Mina,e, Jeremy Hensonf,g, Shaillay Kumar Dograh, Sung H. Hwangi, Bruce D. Hammocki, Lihua J. Zhuj, Roger R. Reddelf,g, W. Mark Saltzmanc, Robert H. Weissb,k, Sandy Changa,e, Michael R. Greenl,1, and Narendra Wajapeyeea,1 Departments of aPathology and eLaboratory Medicine, Yale University School of Medicine, New Haven, CT 06510; iDepartment of Entomology and bDivision of Nephrology, Department of Internal Medicine, University of California, Davis, California 95616; Departments of cBiomedical Engineering and dMolecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511; fSydney Medical School, University of Sydney, NSW 2006, Australia; gCancer Research Unit, Children’s Medical Research Institute, Westmead, NSW 2145, Australia; hSingapore Institute of Clinical Sciences, Agency for Science Technology and Research (A*STAR), Brenner Center for Molecular Medicine, Singapore 117609; lHoward Hughes Medical Institute and jPrograms in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Massachusetts 01605; and kDepartment of Medicine, Mather VA Medical Center, Sacramento, CA 9565 Contributed by Michael R. Green, June 19, 2014 (sent for review June 8, 2014) Tumor suppressor p53 plays an important role in mediating growth dition to its role in cell cycle regulation, p21 has been shown in inhibition upon telomere dysfunction. Here, we show that loss of a variety of studies to repress apoptosis (9–13). the p53 target gene cyclin-dependent kinase inhibitor 1A (CDKN1A, Here,westudytheroleofp21inthe context of telomerase in- also known as p21WAF1/CIP1) increases apoptosis induction following hibition.
    [Show full text]
  • Full Text (PDF)
    ResearchArticle DNA Damage–Dependent Translocation of B23 and p19ARF Is Regulated by the Jun N-Terminal Kinase Pathway Orli Yogev,1 Keren Saadon,1 Shira Anzi,1 Kazushi Inoue,2 and Eitan Shaulian1 1Department of Experimental Medicine and Cancer Research, Hebrew University Medical School, Jerusalem, Israel and 2Departments of Pathology/Cancer Biology, Wake Forest University Health Sciences, Winston-Salem, North Carolina Abstract arrest or apoptosis. However, increased tumor development in The dynamic behavior of the nucleolus plays a role in the triple knockout mice nullizygous for ARF, p53, and Mdm2 showed detection of and response to DNA damage of cells. Two that ARF acts also in a p53-independent manner (11). Some of the nucleolar proteins, p14ARF/p19ARF and B23, were shown to p53-independent activity is attributed to its ability to reduce rRNA translocate out of the nucleolus after exposure of cells to DNA- processing (12, 13) and inhibit oncogene-induced transcription damaging agents. This translocation affects multiple cellular (14, 15). functions, such as DNA repair, proliferation, and survival. In ARF augments p53 activity mainly in response to oncogenic this study, we identify a pathway and scrutinize the mecha- stress. Its expression is up-regulated in response to deregulated nisms leading to the translocation of these proteins after oncogenic activity due to elevated transcription governed by several transcription factors, such as E2F (16), c-myc (17), AP-1 exposure of cells to DNA-damaging agents. We show that redistribution of B23 and p19ARF after the exposure to (18), or by oncogenic Ras (19). A second mechanism regulating ARF activity involves the control of its subcellular localization (3).
    [Show full text]
  • P14ARF Inhibits Human Glioblastoma–Induced Angiogenesis by Upregulating the Expression of TIMP3
    P14ARF inhibits human glioblastoma–induced angiogenesis by upregulating the expression of TIMP3 Abdessamad Zerrouqi, … , Daniel J. Brat, Erwin G. Van Meir J Clin Invest. 2012;122(4):1283-1295. https://doi.org/10.1172/JCI38596. Research Article Oncology Malignant gliomas are the most common and the most lethal primary brain tumors in adults. Among malignant gliomas, 60%–80% show loss of P14ARF tumor suppressor activity due to somatic alterations of the INK4A/ARF genetic locus. The tumor suppressor activity of P14ARF is in part a result of its ability to prevent the degradation of P53 by binding to and sequestering HDM2. However, the subsequent finding of P14ARF loss in conjunction with TP53 gene loss in some tumors suggests the protein may have other P53-independent tumor suppressor functions. Here, we report what we believe to be a novel tumor suppressor function for P14ARF as an inhibitor of tumor-induced angiogenesis. We found that P14ARF mediates antiangiogenic effects by upregulating expression of tissue inhibitor of metalloproteinase–3 (TIMP3) in a P53-independent fashion. Mechanistically, this regulation occurred at the gene transcription level and was controlled by HDM2-SP1 interplay, where P14ARF relieved a dominant negative interaction of HDM2 with SP1. P14ARF-induced expression of TIMP3 inhibited endothelial cell migration and vessel formation in response to angiogenic stimuli produced by cancer cells. The discovery of this angiogenesis regulatory pathway may provide new insights into P53-independent P14ARF tumor-suppressive mechanisms that have implications for the development of novel therapies directed at tumors and other diseases characterized by vascular pathology. Find the latest version: https://jci.me/38596/pdf Research article P14ARF inhibits human glioblastoma–induced angiogenesis by upregulating the expression of TIMP3 Abdessamad Zerrouqi,1 Beata Pyrzynska,1,2 Maria Febbraio,3 Daniel J.
    [Show full text]
  • Loss of P21 Disrupts P14arf-Induced G1 Cell Cycle Arrest but Augments P14arf-Induced Apoptosis in Human Carcinoma Cells
    Oncogene (2005) 24, 4114–4128 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc Loss of p21 disrupts p14ARF-induced G1 cell cycle arrest but augments p14ARF-induced apoptosis in human carcinoma cells Philipp G Hemmati1,3, Guillaume Normand1,3, Berlinda Verdoodt1, Clarissa von Haefen1, Anne Hasenja¨ ger1, DilekGu¨ ner1, Jana Wendt1, Bernd Do¨ rken1,2 and Peter T Daniel*,1,2 1Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charite´, Campus Berlin-Buch, Berlin-Buch, Germany; 2Max-Delbru¨ck-Center for Molecular Medicine, Berlin-Buch, Germany The human INK4a locus encodes two structurally p16INK4a and p14ARF (termed p19ARF in the mouse), latter unrelated tumor suppressor proteins, p16INK4a and p14ARF of which is transcribed in an Alternative Reading Frame (p19ARF in the mouse), which are frequently inactivated in from a separate exon 1b (Duro et al., 1995; Mao et al., human cancer. Both the proapoptotic and cell cycle- 1995; Quelle et al., 1995; Stone et al., 1995). P14ARF is regulatory functions of p14ARF were initially proposed to usually expressed at low levels, but rapid upregulation be strictly dependent on a functional p53/mdm-2 tumor of p14ARF is triggered by various stimuli, that is, suppressor pathway. However, a number of recent reports the expression of cellular or viral oncogenes including have implicated p53-independent mechanisms in the E2F-1, E1A, c-myc, ras, and v-abl (de Stanchina et al., regulation of cell cycle arrest and apoptosis induction by 1998; Palmero et al., 1998; Radfar et al., 1998; Zindy p14ARF. Here, we show that the G1 cell cycle arrest et al., 1998).
    [Show full text]
  • Alterations of P14arf, P53, and P73 Genes Involved in the E2F-1-Mediated Apoptotic Pathways in Non-Small Cell Lung Carcinoma
    [CANCER RESEARCH 61, 5636–5643, July 15, 2001] Alterations of p14ARF, p53, and p73 Genes Involved in the E2F-1-mediated Apoptotic Pathways in Non-Small Cell Lung Carcinoma Siobhan A. Nicholson,1 Nader T. Okby,1 Mohammed A. Khan, Judith A. Welsh, Mary G. McMenamin, William D. Travis, James R. Jett, Henry D. Tazelaar, Victor Trastek, Peter C. Pairolero, Paul G. Corn, James G. Herman, Lance A. Liotta, Neil E. Caporaso, and Curtis C. Harris2 Laboratory of Human Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892 [S. A. N., M. A. K., J. A. W., M. G. M., L. A. L., N. E. C., C. C. H.]; Orange Pathology Associates, Middleton, New York 10940 [N. T. O.]; Armed Forces Institute of Pathology, Washington, DC 20306 [S. A. N., W. D. T.]; Mayo Clinic, Rochester, Minnesota 55905 [J. R. J., H. D. T., V. T., P. C. P.]; and The Johns Hopkins Oncology Center, Baltimore, Maryland 21231 [P. G. C., J. G. H.] ABSTRACT encoded by a separate exon 1␤ that lies ϳ20 kb upstream of exon 1␣ and shares exons 2 and 3 as read in an ARF, giving rise to a protein ARF Overexpression of E2F-1 induces apoptosis by both a p14 -p53- and INK4a ARF completely unrelated to p16 (14). Despite its unrelated structure, a p73-mediated pathway. p14 is the alternate tumor suppressor prod- ARF p14 also is capable of causing cell cycle arrest in G1 and G2. uct of the INK4a/ARF locus that is inactivated frequently in lung carci- ARF nogenesis. Because p14ARF stabilizes p53, it has been proposed that the loss p14 binds to and antagonizes the actions of MDM2, a negative of p14ARF is functionally equivalent to a p53 mutation.
    [Show full text]
  • Expression of P16 INK4A and P14 ARF in Hematological Malignancies
    Leukemia (1999) 13, 1760–1769 1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu Expression of p16INK4A and p14ARF in hematological malignancies T Taniguchi1, N Chikatsu1, S Takahashi2, A Fujita3, K Uchimaru4, S Asano5, T Fujita1 and T Motokura1 1Fourth Department of Internal Medicine, University of Tokyo, School of Medicine; 2Division of Clinical Oncology, Cancer Chemotherapy Center, Cancer Institute Hospital; 3Department of Hematology, Showa General Hospital; 4Third Department of Internal Medicine, Teikyo University, School of Medicine; and 5Department of Hematology/Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan The INK4A/ARF locus yields two tumor suppressors, p16INK4A tumor suppressor genes.10,11 In human hematological malig- ARF and p14 , and is frequently deleted in human tumors. We nancies, their inactivation occurs mainly by means of homo- studied their mRNA expressions in 41 hematopoietic cell lines and in 137 patients with hematological malignancies; we used zygous deletion or promoter region hypermethylation a quantitative reverse transcription-PCR assay. Normal periph- (reviewed in Ref. 12). In tumors such as pancreatic adenocar- eral bloods, bone marrow and lymph nodes expressed little or cinomas, esophageal squamous cell carcinomas and familial undetectable p16INK4A and p14ARF mRNAs, which were readily melanomas, p16INK4A is often inactivated by point mutation, detected in 12 and 17 of 41 cell lines, respectively. Patients with 12 INK4A which is not the case in hematological malignancies. On hematological malignancies frequently lacked p16 INK4C INK4D ARF the other hand, genetic aberrations of p18 or p19 expression (60/137) and lost p14 expression less frequently 12 (19/137, 13.9%).
    [Show full text]
  • Human P14arf-Mediated Cell Cycle Arrest Strictly Depends on Intact P53 Signaling Pathways
    Oncogene (2002) 21, 3207 ± 3212 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Human p14ARF-mediated cell cycle arrest strictly depends on intact p53 signaling pathways H Oliver Weber1,2, Temesgen Samuel1,3, Pia Rauch1 and Jens Oliver Funk*,1 1Laboratory of Molecular Tumor Biology, Department of Dermatology, University of Erlangen-Nuremberg, 91052 Erlangen, Germany; 2Regulation of Cell Growth Laboratory, National Cancer Institute, Frederick, Maryland, MD 21702-1201, USA The tumor suppressor ARF is transcribed from the INK4a/ 4 and 6 activities, thus leading to decreased phosphor- ARF locus in partly overlapping reading frames with the ylation of RB and to G1 arrest. Cells that are de®cient CDK inhibitor p16Ink4a. ARF is able to antagonize the for RB are resistant to p16Ink4a-mediated cell cycle MDM2-mediated ubiquitination and degradation of p53, arrest (Sherr and Roberts, 1995; Weinberg, 1995). leading to either cell cycle arrest or apoptosis, depending on ARF is also a cell cyle inhibitor. It does not directly the cellular context. However, recent data point to inhibit CDKs but interferes with the function of additional p53-independent functions of mouse p19ARF. MDM2 to destabilize p53. ARF may be activated by Little is known about the dependency of human p14ARF aberrant activation of oncoproteins such as Ras function on p53 and its downstream genes. Therefore, we (Palmero et al., 1998), c-myc (Zindy et al., 1998), analysed the mechanism of p14ARF-induced cell cycle arrest E1A (de Stanchina et al., 1998), Abl (Radfar et al., in several human cell types.
    [Show full text]
  • P14arf Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional P53
    [CANCER RESEARCH 64, 4338–4345, June 15, 2004] p14ARF Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53 Pellegrino G. Magro,1,2 Angelo J. Russo,1,2 Wei-Wei Li,2 Debabrata Banerjee,3 and Joseph R. Bertino3 1Joan and Sanford I. Weill Graduate School of Medical Sciences of Cornell University, New York, New York; 2Memorial Sloan Kettering Cancer Center, New York, New York; and 3The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey ABSTRACT Although much is known about the function of ARF in the p53 pathway, less is known about its functions in human tumor growth and ARF The p14 protein, the product of an alternate reading frame of the chemosensitivity independently of its regulation of p53 via binding to INK4A/ARF locus on human chromosome 9p21, disrupts the ability of MDM2. Mouse embryonic fibroblasts expressing E1A and exogenous MDM2 to target p53 for proteosomal degradation and causes an increase ARF are more sensitive to killing by doxorubicin than their normal in steady-state p53 levels, leading to a G1 and G2 arrest of cells in the cell cycle. Although much is known about the function of p14ARF in the p53 counterparts that only express E1A (7). Alterations in the INK4A/ pathway, not as much is known about its function in human tumor growth ARF locus reduce p53 induction and cytotoxicity of cyclophospha- and chemosensitivity independently of up-regulation of p53 protein levels. mide in mouse lymphomas (16). Also, recombinant adenovirus-me- To learn more about its effect on cellular proliferation and chemoresis- diated p14ARF overexpression sensitizes MCF-7 breast cancer cells to tance independent of p53 up-regulation, human HT-1080 fibrosarcoma cisplatin (17).
    [Show full text]
  • Differential Roles of P16 and P14 Genes in Prognosis of Oral
    414 Differential Roles of p16INK4A and p14ARF Genes in Prognosis of Oral Carcinoma R. Sailasree,1 A. Abhilash,1 K.M. Sathyan,1 K.R. Nalinakumari,2 Shaji Thomas,3 and S. Kannan1 1Laboratory of Cell Cycle Regulation and Molecular Oncology, Division of Cancer Research, 2Division of Dental Surgery, and 3Division of Surgical Oncology, Regional Cancer Center, Thiruvananthapuram, Kerala, India Abstract INK4A Background: Oral cancer patients are found to have observed in 30% of the cases. p16 deletion was poor clinical outcome and high disease recurrence associated with aggressive tumors, as evidenced by the rate, in spite of an aggressive treatment regimen. The nodal involvement of the disease. Low or absence of inactivation of INK4A/ARF loci is reported to be second p16INK4A protein adversely affected the initial treat- INK4A to p53 inactivation in human cancers. The purpose of ment response. Promoter methylation of p16 was this study was to assess the prognostic significance of associated with increased disease recurrence and acts the molecular aberrations in the INK4A locus for as an independent predictor for worse prognosis. ARF effective identification of aggressive oral carcinoma Surprisingly, p14 methylation associated with cases needing alternate therapy. lower recurrence rate in oral cancer patients with a Materials and Methods: The study composed of 116 good clinical outcome. Overall survival of these patients freshly diagnosed with oral carcinoma. The patients was associated with tumor size, nodal disease, INK4A genetic and epigenetic status of the p16 and and p16INK4A protein expression pattern. Our results ARF INK4A ARF p14 genes was evaluated. The relation between indicate that p16 and p14 alterations constitute these genic alterations and different treatment end a major molecular abnormality in oral cancer cases.
    [Show full text]
  • ARF Promotes Accumulation of Retinoblastoma Protein Through Inhibition of MDM2
    Oncogene (2007) 26, 4627–4634 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE ARF promotes accumulation of retinoblastoma protein through inhibition of MDM2 DLF Chang1, W Qiu1, H Ying2, Y Zhang3, C-Y Chen4 and Z-XJ Xiao1,2 1Graduate Program in Molecular Medicine, Department of Medicine, Boston University School of Medicine, Boston, MA, USA; 2Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA; 3The Pulmonary Center, Boston University School of Medicine, Boston, MA, USA and 4Department of Pathology, Boston University School of Medicine, Boston, MA, USA The INK4a/ARF locus,encoding two tumor suppressor binds to and inhibits cyclin D-associated kinases proteins,p16 INK4a and p14ARF (ARF),plays key roles in resulting in hypophosphorylated retinoblastoma protein many cellular processes including cell proliferation, (Rb), a biologically active Rb species in tumor suppres- apoptosis,cellular senescence and differentiation. Inacti- sion (Weinberg, 1995). It was later discovered that the vation of INK4a/ARF is one of the most frequent events same locus encodes a second protein through the usage during human cancer development. Although p16INK4a is of a separate promoter with a distinct first exon (exon a critical component in retinoblastoma protein (Rb)- 1b), which results in translation from an alternate mediated growth regulatory pathway,p14 ARF plays a reading frame (ARF) encoding a basic, nucleolar pivotal role in the activation of p53 upon oncogenic stress protein named p14ARF in human (p19ARF in mouse). signals. A body of evidence indicates that ARF also ARF shares no amino acid homology with p16INK4a possesses growth suppression functions independent of protein and is activated by oncogenic stress signals, such p53,the mechanism of which is not well understood.
    [Show full text]
  • Regulation of the P53 Family Proteins by the Ubiquitin Proteasomal Pathway
    International Journal of Molecular Sciences Review Regulation of the p53 Family Proteins by the Ubiquitin Proteasomal Pathway Scott Bang, Sandeep Kaur and Manabu Kurokawa * Department of Biological Sciences, Kent State University, Kent, OH 44242, USA; [email protected] (S.B.); [email protected] (S.K.) * Correspondence: [email protected]; Tel.: +1-330-672-2979 Received: 27 November 2019; Accepted: 24 December 2019; Published: 30 December 2019 Abstract: The tumor suppressor p53 and its homologues, p63 and p73, play a pivotal role in the regulation of the DNA damage response, cellular homeostasis, development, aging, and metabolism. A number of mouse studies have shown that a genetic defect in the p53 family could lead to spontaneous tumor development, embryonic lethality, or severe tissue abnormality, indicating that the activity of the p53 family must be tightly regulated to maintain normal cellular functions. While the p53 family members are regulated at the level of gene expression as well as post-translational modification, they are also controlled at the level of protein stability through the ubiquitin proteasomal pathway. Over the last 20 years, many ubiquitin E3 ligases have been discovered that directly promote protein degradation of p53, p63, and p73 in vitro and in vivo. Here, we provide an overview of such E3 ligases and discuss their roles and functions. Keywords: apoptosis; cancer; Tp53; Tp63; Tp73; ubiquitination; E3 ligase 1. Introduction The “guardian of the genome”, p53, has long been known to regulate the cellular responses of DNA repair, cell senescence, cell cycle arrest, and apoptosis [1]. Mice deficient in p53 exhibit significantly increased susceptibility to tumor formation compared to wild type mice and are a valuable tool with which to study the effects of p53 on tumor initiation and progression [2].
    [Show full text]
  • The H3k27me3 Demethylase JMJD3 Contributes to the Activation of the INK4A−ARF Locus in Response to Oncogene- and Stress-Induced Senescence
    Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION teins leads to increased H3K27me3 levels and silencing of The H3K27me3 demethylase the INK4A–ARF locus. The PcG proteins are frequently JMJD3 contributes to the overexpressed in human tumors, and their contribution to proliferation is ascribed primarily to their role in re- activation of the INK4A–ARF gulating the expression of the INK4A–ARF locus (Jacobs et al. 1999; Bracken et al. 2007; Dietrich et al. 2007). locus in response to oncogene- Recently, we and others identified two histone lysine and stress-induced senescence demethylases—JMJD3 and UTX—that specifically cata- lyze the demethylation of di- and trimethylated H3K27 Karl Agger, Paul A.C. Cloos, Lise Rudkjær, (H3K27me2/me3) (Agger et al. 2007; De Santa et al. 2007; Kristine Williams, Gitte Andersen, Jepsen et al. 2007; Lan et al. 2007; Lee et al. 2007). Jesper Christensen, and Kristian Helin1 We wanted to understand whether one or both of these enzymes could be involved in the active removal of Biotech Research and Innovation Centre (BRIC) and Centre for H3K27me3 from the INK4A–ARF locus during induction Epigenetics, University of Copenhagen, DK-2200 Copenhagen, of stress-induced senescence and oncogene-induced se- Denmark nescence (OIS). The tumor suppressor proteins p16INK4A and p14ARF, encoded by the INK4A–ARF locus, are key regulators of Results and Discussion cellular senescence. The locus is epigenetically silenced by the repressive H3K27me3 mark in normally growing JMJD3 expression is induced by activation cells, but becomes activated in response to oncogenic of the RAS–RAF pathway stress.
    [Show full text]