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University Micrailms International 300 N CYTOGENETIC ABNORMALITIES AND THE PROGRESSION TO INVASION IN A375P HUMAN MELANOMA CELLS IN VITRO Item Type text; Thesis-Reproduction (electronic) Authors Greeff, Christopher Whitney, 1961- Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 27/09/2021 11:46:32 Link to Item http://hdl.handle.net/10150/276462 INFORMATION TO USERS This reproduction was made from a copy of a document sent to us for microfilming. While the most advanced technology has been used to photograph and reproduce this document, the quality of the reproduction is heavily dependent upon the quality of the material submitted. The following explanation of techniques is provided to help clarify markings or notations which may appear on this reproduction. 1.The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure complete continuity. 2. When an image on the film is obliterated with a round black mark, it is an indication of either blurred copy because of movement during exposure, duplicate copy, or copyrighted materials that should not have been filmed. For blurred pages, a good image of the page can be found in the adjacent frame. If copyrighted materials were deleted, a target note will appear listing the pages in the adjacent frame. 3. When a map, drawing or chart, etc., is part of the material being photographed, a definite method of "sectioning" the material has been followed. It is customary to begin filming at the upper left hand corner of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning is continued again-beginning below the first row and continuing on until complete. 4. For illustrations that cannot be satisfactorily reproduced by xerographic means, photographic prints can be purchased at additional cost and inserted into your xerographic copy. These prints are available upon request from the Dissertations Customer Services Department. 5. Some pages in any document may have indistinct print. In all cases the best available copy has been filmed. University Micrailms International 300 N. Zeeb Road Ann Arbor, Ml 48106 Order Number 1331403 Cytogenetic abnormalities and the progression to invasion in A375P human melanoma cells in vitro Greeff, Christopher Whitney, M.S. The University of Arizona, 1987 UMI 300 N. ZeebRd. Ann Arbor, MI 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V . 1. Glossy photographs or pages 2. Colored illustrations, paper or print 3. Photographs with dark background ^ 4. Illustrations are poor copy 5. Pages with black marks, not original copy s/ 6. Print shows through as there is text on both sides of page 7. Indistinct, broken or small print on several pages v 8. Print exceeds margin requirements 9. Tightly bound copy with print lost in spine 10. Computer printout pages with indistinct print 11. Page(s) lacking when material received, and not available from school or author. 12. Page(s) seem to be missing in numbering only as text follows. 13. Two pages numbered . Text follows. 14. Curling and wrinkled pages 15. Dissertation contains pages with print at a slant, filmed as received 16. Other University Microfilms International CYTOGENETIC ABNORMALITIES AND THE PROGRESSION TO INVASION IN A375P HUMAN MELANOMA CELLS IN VITRO by Christopher Whitney Greeff A Thesis Submitted to the Faculty of the DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE WITH A MAJOR IN MICROBIOLOGY In the Graduate College THE UNIVERSITY OF ARIZONA 19 8 7 STATEMENT BY AUTHOR This thesis has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowl­ edgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. APPROVAL BY THESIS DIRECTOR This thesis has been approved on the date shown below: 7V.S.C MeJU* M. J. C. HENDRIX Date Professor of Anatomy ACKNOWLEDGMENTS I would like to gratefully acknowledge the help and invaluable assistance of the following individuals, without whom the completion of this work would not have been possible. Dr. J. Trent and Iris Veomett, for their guidance in performing cytogenetic procedures and analysis. Dr. 0. Ward, for his thoughtful help. Sandra J. Bevacqua, for her assistance in the identification of double minute chromosomes and for the cell lines used in that endeavor, as well as her effort in the compilation and presentation of the double minute data. Matthew J. Bullock, for the arduous work involved in determining the degree of aneuploidy for each cell line. Elizabeth Seftor, for her unselfish contributions in the laboratory cell culture of all cell lines described in this work. iii TABLE OP CONTENTS Page LIST OF ILLUSTRATIONS v LIST OF TABLES vi ABSTRACT vii CHAPTER 1. INTRODUCTION 1 2. MATERIALS AND METHODS 26 Cell Lines 26 Cell Culture 28 Mega-MICS Assembly and Operation .... 29 Cytogenetic Analysis 32 3. RESULTS 37 Karyotyping 37 Double Minute Chromosomes 55 4. DISCUSSION 60 LIST OF REFERENCES 69 iv LIST OF ILLUSTRATIONS Figure Page 1. Mega-MICS Chambers 30 2. Aneuploidy in A375P P33 38 3. Aneuploidy in A375P-1 39 4. Aneuploidy in A375P P37 3& 5. Aneuploidy in A375P-2 40 6. Aneuploidy in A375P P41 40 7. Aneuploidy in A375P-3,. 42 8. Aneuploidy in A375P P55 42 9. Karyotype of A375P P33 (Parental Cell Line) . 43 10. Karyotype of A375P P33 (Parental Cell Line) . 44 11. Karyotype of A375P-1 45 12. Karyotype of A375P P37 47 13. Karyotype of A375P-1 48 14. Karyotype of A375P-2 49 15. Karyotype of A375P-2 50 16. Karyotype of A375P P41 52 17. Karyotype of A375P-3 53 18. Karyotype of A375P-3 54 19. Karyotype of A375P P55 56 20. Loss of Double Minutes with Tissue Culture Passage 59 v LIST OF TABLES Table Page 1. Quantitation of Double Minute Chromosomes ... 57 vi ABSTRACT A study was undertaken to determine whether cytogenetic abnormalities can be identified in an invasive melanoma cell population that has been selected in vitro out of a larger cell population of low invasive potential. The selecting agent was a denuded human amniotic membrane situated within Mega-Membrane Invasion Culture System chambers. Invasive cells were collected, grown, and harvested for cytogenetic analysis. Metaphases of these cells were examined for chromosomal abnormalities and for evidence of gene amplification in the form of double minute chromosomes. Invasive cell lines evinced changes in their degree of aneuploidy which were not seen in parental control lines of the same passage number. Significant karyotypic abnormalities identified in invasive cell lines were an increased dosage of chromosome 7 and multiple lq translocation marker chromosomes. Double minute chromosomes were found in up to 18% of invasive cell metaphases examined and in 3% of parental controls. The incidence of double minutes was found to decrease as a function of passage number. vii CHAPTER 1 INTRODUCTION Malignant melanoma is presently one of the most severe and painful neoplastic diseases known, and it is widely recognized to be increasing in incidence across the globe. This increase may be attributable both to atmos­ pheric ozone degradation and the increasing aesthetic value of a tan among Western Caucasian populations. Whatever the societal and environmental etiology of melanoma may be, alarmingly little is known about the fundamental biology and genetics of melanocytes and their malignant counter­ parts, and in the absence of such specific knowledge clinicians are forced to employ relatively nonspecific therapeutic regimes in the treatment of this malignant dis­ ease. Melanocytes are an ubiquitous cellular component of the pigmentary system of the skin, and the dark brown pig­ ment melanin can be found in such histologically diverse sites as the substantia nigra in the midbrain and the reti­ nal pigment epithelium of the eye (1). The embryologic origin of melanocytes is in the neural crest, and they are thought to invade the epidermis around the third month of 1 2 fetal development. In the skin melanocytes can be found in the stratum basal of the epidermis and in the underlying layers of the dermis, where they are generally seen to have a branching morphology. Most of the pigmented cells of the skin are actually keratinocytes which contain melanosomes transferred to them from melanocytes via the process of cytocrine secretion (1). It is important to note here that melanin is only elaborated in melanocytes, which may be distinguished biochemically from melanin-containing kera­ tinocytes by the presence of the enzyme tyrosinase. Tyro­ sinase utilizes a copper ion (Cu++) to catalyze the oxidation of tyrosine to dihyroxyphenylalanine (Dopa) and Dopa quinone, both of which can subsequently oxidize to form the red pigment Hallochrome.
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