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Isolation of a cell surface antigen specific for human acute myelogenous leukemia cells Shipman, Robert Charles 1982

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C-l.  ISOLATION SPECIFIC  OP FOR  A CELL  SURFACE  HUMAN  ACUTE  LEUKEMIA  CELLS  ANTIGEN  MYELOGENOUS  by ROBERT C H A R L E S B.Sc,  University  A THESIS  of B r i t i s h  SUBMITTED  THE  SHIPMAN Columbia,  IN P A R T I A L  REQUIREMENTS MASTER  FOR  OF  FULFILLMENT  THE  DEGREE  OF  SCIENCE  i n THE  FACULTY  OF  (Department  We  accept to  THE  this  GRADUATE of  the r e q u i r e d  UNIVERSITY  OF  Robert  as  Charles  conforming  standard  BRITISH  August 0  STUDIES  Microbiology)  thesis  1980  COLUMBIA  1982 Shipman,  1982  OF  In presenting  this thesis  i n partial  r e q u i r e m e n t s f o r an a d v a n c e d  fulfilment of the  degree a t t h e U n i v e r s i t y  of  B r i t i s h Columbia, I agree that  the Library  it  f r e e l y a v a i l a b l e f o r r e f e r e n c e and study.  shall I  make  further  agree t h a t permission f o r e x t e n s i v e copying o f t h i s  thesis  f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my department  o r by h i s o r h e r r e p r e s e n t a t i v e s .  understood that  copying or p u b l i c a t i o n of t h i s  f o r f i n a n c i a l gain  of  H\CROB\0LOG-V  The U n i v e r s i t y o f B r i t i s h 1956 Main M a l l V a n c o u v e r , Canada V6T 1Y3 Date  thesis  s h a l l n o t be a l l o w e d w i t h o u t my  permission.  Department  It i s  SEPT. 3 . , 9 8 2  Columbia  written  ABSTRACT  Methods and  are described  #3), a p p a r e n t l y  myelogenous acrylamide  demonstrated cell did  with  of normal  proliferative  of  totally  comparable  dalton  (anti-AML#1,  membrane  preparations.  detectable, aggregates the  cell  was  shown  phoresis  only  o f AML  membrane  material  band#1  with  of  inhibiting  promyelocytic  band#1  whereas  demonstrate  AML  line  AML  membranes  by  a s AML  bands#2 and  AML  two-dimensional  was  also  o f anti-AML#1  inhibition.  normal  capable  cell  three  cell  were  examined was  represented  band#1  PAGE a n d material  gel electroa n d a mean  t o be  antiserum  HL-60, as d e m o n s t r a t e d from  AML  daltons  shown  AML  of a l l  band#1 #3  from  that  was  and  lympho-  of non-reducing  o f 68,000  AML  with  showed  absorbed  poly-  proteins  preparations  material  procedures.  material  material  this  cell  acute  with  i n the ELISA,  with  #2  f o r absorbed  of p a t i e n t s  identified  weight  the r e a c t i o n cell  a l l three  membrane  (#1,  non-reducing  of patients  weight)  #3)  preparative  a molecular  AML  cells  or a r t i f a c t s  t o be h o m o g e n o u s  o f 7.16.  not  When  i n d i c a t i n g that  pi  analysis,  and  with  i n the ELISA,  the r e a c t i o n , #2  on  Inhibition studies  molecular  inhibiting  SDS-PAGE,  against  cell  bands  of patients  i n d i v i d u a l s or those  antisera  in  from  protein  isolated  specificity,  disorders.  (68,000  were  raised  preparations  not r e a c t  band#1  Antisera  three  to c e l l s  (AML),  absolute  membrane  cells  unique  leukemia gels.  whereby  capable  with  by F A C S  membranes  the IV  could  i i i  TABLE  Abstract Table  OF  CONTENTS  i i  of  Contents  i i i  List  of  Tables..  iv  List  of  Figures  v  List  of Abbreviations  Acknowledgements Introduction Materials  and  vi v i i 1  Methods  9  Results  19  Discussion  43  References  46  i v  LIST  Table  I.  OF  TABLES  Title  R e s u l t s o f FACS I V a n a l y s i s o f HL-60 c e l l s with a n t i s e r a p r e - i n c u b a t e d w i t h AML b a n d # 1 , absorbed n o r m a l human c e l l membrane a n t i g e n o r PBS p r i o r to l a b e l l i n g and a n a l y s i s .  Page  38  V  LIST  Figure  1A.  1.  2.  3.  4.  5.  6.  7.  8.  9.  OF F I G U R E S  Title  Page  Ontogeny of the c e l l l i n e a g e s i n v o l v e d i n human a c u t e m y e l o g e n o u s l e u k e m i a (AML) a n d o t h e r h e m a t o p o e t i c stem c e l l d i s o r d e r s .  6  ELISA o f i n d i v i d u a l e x t r a c t s of e i t h e r AML or n o r m a l human p e r i p h e r a l b l o o d leucocytes (PBL) w i t h a b s o r b e d a n t i - A M L a n t i s e r u m .  21  E L I S A o f e x t r a c t s f r o m AML a n d v a r i o u s b o n e marrow c e l l s a m p l e s w i t h e i t h e r a n t i - A M L o r a n t i - n o r m a l human a n t i s e r u m .  23  A n a l y t i c a l non-reducing g e l p a t t e r n from membrane e x t r a c t s o f n o r m a l human PBL a n d b l a s t c e l l s f r o m a n AML p a t i e n t .  26  SDS-PAGE extracts  29  o f a number o f a b s o r b e d membrane from a v a r i e t y o f c e l l s o u r c e s .  S p e c i f i c i t y of v a r i o u s anti-AML a n t i s e r a f o r a b s o r b e d AML e x t r a c t s i n t h e E L I S A .  32  Inhibition ELISA w i t h band#1.  34  of anti-AML a n t i s e r a i n the v a r i o u s c o n c e n t r a t i o n s o f AML  Two-dimensional o f AML b a n d # 1 .  polyacrylamide  gel profile 36  I n h i b i t i o n a n a l y s i s o f anti-AML#1 and a n t i n o r m a l human a n t i s e r a w i t h AML band#1 i n the FACS.  40  I n h i b i t i o n a n a l y s i s o f anti-AML#1 and a n t i n o r m a l human a n t i s e r a w i t h a b s o r b e d n o r m a l human c e l l membrane a n t i g e n i n t h e F A C S .  42  vi  LIST  OF  ABBREVIATIONS  1.  ALL:  Acute  Lymphocytic  Leukemia.  2.  AML:  Acute  Myelogenous  Leukemia.  3.  AMML:  Acute  Monomyelocytic  4.  APML:  Acute  Promyelocytic  5.  CLL:  Chronic Lymphocytic  Leukemia.  6.  CML:  C h r o n i c Myelogenous  Leukemia.  7.  DNA:  Deoxyribonucleic  8.  ELISA:  Enzyme-linked  9.  FACS(IV):  Fluorescence-activated  10.  FITC:  Fluorescein  11.  G-6-PD:  Glucose-6-phoshate  12.  IEF:  Isoelectric  13.  LAA:  Leukemia-associated Antigen.  14.  2-ME:  2-mercaptoethanol.  15.  PAGE:  Polyacrylamide Gel  16.  2D-PAGE:  2-dimensiona1  17.  SDS-PAGE:  Sodium-Dodecyl-Sulphate  Leukemia. Leukemia.  Acid.  Immunosorbent Cell  Assay. Sorter.  I sothiocyanate. Dehydrogenase.  Focussing.  Electrophoresis.  Polyacrylamide Gel  Polyacrylamide Gel  Electrophoresis. 18.  PBS:  Phosphate  Buffered  19.  PBL:  Peripheral  20.  PCV:  Polycythemia  21.  RNA:  Ribonucleic  Blood  Acid.  Saline.  Leucocyte.  Vera.  Electrophoresis.  vi i  ACKNOWLEDGEMENTS  I  would  people  who  complete  like  " helped  this  remaining.  ing  Angus on  my  a p p r e c i a t i o n t o t h e many  t h e way  fellow students  Seifert  front  from  graduate  Levy  still  forproviding  the beginning students  c a n and s p e c i a l  f o r p r o v i d i n g moral  and a b a t i n g  me t o  of sanity  Dr. J u l i a  and g u i d a n c e  t o my  " and e n a b l e d  a semblance  and f o r e m o s t ,  graduate  and K e i t h  t h e home  with  support  Thanks  as o n l y  along  thesis  First  opportunity, project.  to express  this  f o r understandthanks  (morel)  the maelstroms  of  t o Barb support  of confusion  thatarose.  "....For he  afterwards  has s u f f e r e d  what  a man  long  you ask and seek  finds  pleasure  and wandered  long.  t o know." -  Homer.  i n his pains, So  I will  tell  when you  1  INTRODUCTION  In the f i e l d s  o f tumour immunology and tumour v i r o l o g y  t h e r e has been a g r e a t d e a l o f s p e c u l a t i o n on t h e r e l a t e d n e s s o f c a r c i n o g e n e s i s and v i r a l a concommitant  has a l s o been  i n c r e a s e i n t h e amount o f r e s e a r c h d i r e c t e d  toward d e t e r m i n i n g b r i n g about  o n c o g e n e s i s . There  t h e manner by w h i c h o n c o g e n i c v i r u s e s  n e o p l a s t i c t r a n s f o r m a t i o n of normal c e l l s .  Numerous  papers i n the l i t e r a t u r e  have d e a l t w i t h t h e i s o l a t i o n and  cloning  f r o m RNA  o f DNA  neoplastic recipient  sequences  t r a n s f o r m a t i o n when used cells  (1-5).  cloned  transforming  normal  cellular  in  t h e DNA  tumour v i r u s e s w h i c h  silent has  sequences  using  as p r o b e s , the presence of  genes h o m o l o g o u s t o t h e c l o n e d  of normal, n o n - n e o p l a s t i c  cells  i n normal, non-neoplastic  cells  viral  (6).  cellular  transcribed i n neoplastic cells  and  (7).  oncogenes  Subsequent  genes a r e transcriptionally  Other  research  s u g g e s t e d t h a t t h e t a r g e t s f o r t r a n s f o r m a t i o n , i n d u c e d by  v i r u s or c h e m i c a l set of c e l l u l a r  carcinogens,  f r o m human n e o p l a s m s  potential  comprise a s m a l l but d i s c r e t e  oncogenes which a r e p r e s e n t ,  i n normal, non-neoplastic DNA  to t r a n s f e c t normal  Many i n v e s t i g a t o r s have r e p o r t e d ,  s t u d i e s have shown t h a t t h e s e n o r m a l actively  cause  cells  (8).  Experiments using  have d e m o n s t r a t e d  and u l t i m a t e n e o p l a s t i c p h e n o t y p e  DNA o f t h e s e n e o p l a s t i c c e l l s that d i f f e r e n t  neoplasms  but i n a c t i v e , sheared  that the t r a n s f o r m i n g a r e encoded  i n the  (9).Additional findings indicate  carry different  transforming  sequences  2  suggesting for  more  the v a r i e t y  present, the  that  some  protein  o f known  research  products  determination (10).  than  There  viruses  themselves  two  the  proteins  still  present  growth these  time  regulatory  analogous  in  the host  cell  proteins.  of this  regard  involvement causative known  these  process  of oncogenic  about  or i n d i r e c t l y ,  cells.  At  least  but the q u e s t i o n  represent  view a t  normal of  insertion  or  causes  regulatory  of  expression  promoter  an  imbalance  system, the  representing  the f i r s t  step(s)  (6).  (Acute  viruses  Myelogenous or chemical  itself,  Leukemia), the carcinogens  i s unclear.  apart  from  and p h y s i o l o g i c a l c o n s e q u e n c e s .  human  research  has been  conducted  Little  as  is  i t s clinical  manifestations leukemia  by  (11). Other  i n the c e l l  i n leukemiogenesis  the disease  of the cellular  ( 1 0 ) . The a c c e p t e d  by v i r a l  growth  AML  as to  "silent"  The i n a p p r o p r i a t e  imbalance  t o human  agents  success  proteins  event,  feedback  DNA  toward  process  products  transformed  unanswered  i s that  the transformation In  virally  h a d some  activation  consequences in  from  cell  of  to i s o l a t e t r a n s f o r m a t i o n - s p e c i f i c  proteins, precipitated  an  of normally  the host  remains  gene  activated, directly  attempted  l a b o r a t o r i e s have  function  represent  or products  integration into  induced  and a l s o  amount o f s p e c u l a t i o n  viral  or  oncogenes  a certain proteins  i s , at  the i s o l a t i o n  i n the transformation  transcriptionally  have  ( 9 ) . There  toward  cellular  i s responsible  role(s)  genes  researchers  oncogene  neoplasms  directed  of these  has been  or not these  human  being  of their  whether  one c e l l u l a r  The m a j o r i t y  a t the  clinical  of  3  level  and  has  acute  leukemias.  Myelogenous for  AML  dealt  of  r e s e a r c h . CML i n that  a  stem  single  individual, extrinsic  cell  thought  that  insertion  stem  of  to  cell  specific  presence  on  such  next  research with  has  been  The  some  shown  bone  leukemic  clonal  origins  normal  (12).  In  of  lineages  this  are  characteristic  of  a  human  the  well  oncogene, to  an  with  since  from  viral  of  other  very  "leukemic",  from  action  and  originate  chromosomal  derived  cancers,  appears  points  "pre-leukemic"  CML  or  the  (Chronic  starting  are  to  of  CML  than  clonally-derived  the  due  mutation  cellular CML  be  correlates  d i s e a s e s would somatic  human  ofCCML  origin  rather  useful  to  marrow  disorders  the  a  leukemias  characteristic  become  as  cell  a  the  provided  cell  clonal  events,  single  has  stem  theories  chronic  i n the  factor (s).  current  the  has  cells  which  hematopoetic  only  Some  Leukemia)  disease,  genic  with  rare  the  i t is onco-  promoter  acting  on  be  case  the  shown  anomaly  by of  a since  the chromosome  1 22  known  as  the  presence  of  a  (Gd  or  a  Gd ) b  All  cells  and  the  are  most  These  cell cells  single i n the  derived  factors likely  factors,  unknown and by  myeloid  Philadelphia  (13).  stem  well  as  the  glucose-6-phosphate  dehydrogenase  cells  of  G-6-PD h e t e r o z y g o t e s  with  from  the  involved their  leukemic to  i n the  origins  alteration  leukemic cell  ) as  of  i t i s supposed the  (Ph  type  contributing  The  chromosome  that  stem  and  the  cells  cell  their  of  the  occurs  ultimately from  of  the  this  are  i n the of  normal  Ph  well.  still myeloid  the  gives  (13). 1  anomaly  d i s e a s e as  targets  replacemant  p o p u l a t i o n exempt  possess  appearance  cause  i n CML the  stem  CML  stem  normal  rise  growth  to  stem a  control  4  mechanisms  i n t h e bone marrow o f l e u k e m i c  individuals  (14,Figure  1 A) .  In AML several  the o v e r a l l  reports  associated  i n the l i t e r a t u r e  concerning  -  antigens  been d e s c r i b e d  picture i s less clear.  (LAAs) and AML  i n which  T h e r e have leukemia-  t h e AML-LAA has  as a g l y c o p r o t e i n o f 7 5 , 0 0 0 - 8 0 , 0 0 0  molecular  weight  ( 1 5 ) , a g l y c o p r o t e i n o f 400,000  molecular  weight  (16) or c e l l  stage  86,000 and 88,000 d a l t o n m o l e c u l a r raised with  to these  various  the a n t i g e n  their  cell  surface.  been o b s e r v e d  Significant  between t h e s e  (AML, AMML and CML) lymphomas) probably  reflects  lines  dalton  ( 1 7 ) . The a n t i s e r a specific  bearing  a n t i s e r a and c e l l s  have n o t r e v e a l e d  from r e l a t e d  ( A L L , C L L , myelomas and spectrum of  o f common LAAs p r e s e n t  characteristic  on  has a l s o  reactivity  t h e p r e s e n c e o f common stem c e l l  malignant c e l l s .  reactivity  the a n t i g e n  cross-reactivity  d i s o r d e r s . This  antigens  of these  weights  and n o n - r e l a t e d  stem c e l l  differentiation surface  and c e l l  dalton  dependent p r o t e i n s of  p r o t e i n s show h i g h  itself  been  antigens,  on t h e  Although genetic  cell  studies  chromosomal a n o m a l i e s , i n  1  AML  analogous  that defined creating  t o t h e Ph genetic  these  i n CML,  these  s t u d i e s have  events are i n t i m a t e l y involved i n  a n o m a l i e s and p r o b a b l y  represent  agents f o r the d i s e a s e  as w e l l .  experiments, using  from the p r o m y e l o c y t i c  line of  HL-60,  DNA  that t h i s  r e c i p i e n t mouse NIH 3T3 c e l l s  experiment s t r o n g l y supports  causative  I n DNA t r a n s f e c t i o n  i t has been d e m o n s t r a t e d  transforming  implied  the c o n t e n t i o n  leukemia DNA  cell  i s capable  (18). This  t h a t w i t h i n the  5  Figure  1A:  Ontogeny o f t h e c e l l  acute myelogenous leukemia cell  (AML)  lineages  involved  and o t h e r h e m a t o p o e t i c  disorders.  AML:  Acute Myelogenous  CML:  C h r o n i c Myelogenous  ALL:  Acute Lymphocytic  CLL:  Chronic Lymphocytic  PCV:  Polycythemia Vera.  AMML/APML:  i n human  Leukemia. Leukemia. Leukemia. Leukemia.  Acute Mono-/Promyelocytic  Leukemia.  stem  6  MULTIPOTENT STEM CELL (Target f o r CML and AML c a u s a t i v e agents)  LYMPHOID STEM CELL  MYELOID STEM CELL  o o  GRANULOCYTES (Eosinophils, Basophils, Neutrophils) *AML ' ~ *CML *AMML/APML *Granuloma MEGAKARYOCYTES (Platelets) *Platelet deficiency  O  T-LYMPHOCYTES * T - c e l l lymphoma *T-ALL *T-CLL  °b  ERYTHROCYTES (Red blood c e l l s ) *PCV (Polycythemia Vera) Disease  s t a t e s denoted by an a s t e r i s k (*).  B-LYMPHOCYTES * B - c e l l lymphoma *B-ALL *B-CLL  DNA  of leukemic  cells  resides  a gene(s)  responsible for  observed  transforming  ability  and f i n a l  neoplastic  of  cells.  these  partially cell  characterized  surface antigen  titre  antiserum  antiserum absolute whereas cell  this cells  shows  and p e r i p h e r a l  cell  Again,  no r e a c t i v i t y  and s i g n i f i c a n t  reacts  the p e r i p h e r a l  clinical  remission  demonstrating surface presence the Both  of this  normal  expression remission  normal  cells  that  does  that  f o r the onset the gene(s)  this  (20,21).  AML  or When  samples,  marrow a n d CML  unrelated  (22).  i s that  extracts  of c e l l  with  samples  this  An antiserum  o f AML  individual  is significant in  "malignancy  marker"  The o b s e r v a t i o n  cells  on t h e c e l l  high  cells  f o r t h e bone  the event  not represent  When  comparable  not appear  of these  a  membrane  panel  result  cells.  does  imply  o f t h e AML-LAA  responsible showing  results  This  and  I V , i t shows  leucocytes  of this  differentiation  of these  finding  (22).  "marker"  (20).  i s seen  cell  blood  the presence  of putatively  produced  l e u c o c y t e s of both  interesting  in  isolated  individuals  a broader  d i s o r d e r s or normal  with  using  specificity  blood  also  or c e l l  normal  phenotype  t o be a n A M L - s p e c i f i c  o f FACS  i s observed from  have  i n rabbits  cells  IV, u s i n g  antiserum  individuals.  marker  f o r AML  extracts  we  and have  i n the ELISA  no r e a c t i v i t y  i n t h e FACS  time,  appears  (19,20)  specificity  same  what  to this  i s tested  membrane  tested  stem  At the present  the  that the  to interfere i s also  on t h e  with  interesting  causing the  surface  of leukemic  the oncogenic  of leukemiogenesis.  and  event  Apart  responsible for production  from of the  8  AML-LAA i s e x p r e s s e d appear  i n AML, CML and r e m i s s i o n c e l l s  t o be d i f f e r e n t i a t i n g  n o r m a l l y , t h e s e s t u d i e s have  shown AML t o be a d i s e a s e o f c l o n a l o r i g i n malignant With  i n which the  c l o n e i s d e r i v e d from a p l u r i p o t e n t  our i s o l a t i o n  suggesting  o f an AML-LAA and d a t a  t h e r e l a t e d n e s s between v i r a l  from  stem c e l l ( 2 2 ) . tumour  activated oncogene viral  t o r e g a r d t h e AML-LAA as a p r o d u c t normal  cellular  T h e r e i s , a t p r e s e n t , no e v i d e n c e  t h e above s u p p o s i t i o n s . S i n c e so l i t t l e precise cellular  and  virallyrecombinant  c e l l u l a r and  growth  f a c t o r or  t o s u p p o r t any o f  i s known a b o u t t h e  mechanisms i n v o l v e d i n t r a n s f o r m a t i o n and  u l t i m a t e l y what e f f e c t bring  normal  o n c o g e n e s ) o r an a b e r r a n t l y e x p r e s s e d  receptor.  of a  i t is  oncogene, a product of a  (a gene composed o f r e c o m b i n e d  i s e x e r t e d by t h e o n c o g e n e p r o d u c t t o  about t r a n s f o r m a t i o n , q u e s t i o n s r e g a r d i n g the nature  f u n c t i o n o f t h e AML-LAA abound. I s t h e AML-LAA a  growth  virology  t r a n s f o r m a t i o n and  the i n d u c t i o n df t r a n s f o r m a t i o n - s p e c i f i c p r o t e i n s , tempting  which  r e g u l a t o r y gene(s)  overproduced  product?  or i n a p p r o p r i a t e l y  Is t h i s gene(s)  expressed  Does e x p r e s s i o n o f t h e AML-LAA u l t i m a t e l y  normal  product  i n leukemic result  i n leukemic  t r a n s f o r m a t i o n ? A n s w e r s t o t h e above q u e s t i o n s a r e n o t immediately research.  cells?  a v a i l a b l e and a r e t h e s u b j e c t s o f o n g o i n g  9  MATERIALS  Preparation  of  7 1x10 cell 30  on  a  AML  the  volumes  by  and  on  sample  min.  normal  ice at  thawing.  from  or  the  Cell  at  400xg  Membrane  and  s o n i c a t e d as  KC1  protein  and  to  a  by  the  supernatent  were  antiserum  Feedback  antiserum;  Antiserum  as  was  in a  the  prepared  extracts.  antigen,  also  and  Rabbits  50%  10 by  sample  of  30%  Cell  pH  Lowry  tested  of  et  minimal  7.2,  0.01M  extracted in then or  were  and  the  total  supernatent  a l . (23). reactive  was  sonication  i n PBS and  50,000xg  Both  the  material  were pellet  using  (24).  a g a i nst  emulsion  ml  at  were  in  Pellets  pellet  for  ELISA  were  PBS.  following  centrifugation  sample  following  5.0  the  or  min,  intensity  membranes  membrane  against  2-3  300)  by  resuspended  min  prepare  for  centrifugation  cell  of  to  probe  removed  min.  were  180  both of  used  d e s c r i b e d above  volume  method  specific  membrane  for  c o n c e n t r a t i o n of  determined  for  The  dialysis  final  cell  buffered saline,  4°C.  50,000xg  extraction  resuspended  and  at  at  were  Dismembrator(Model  pellets  phosphate)  overnight  the  setting  supernatents  (phosphate  centrifuged  a  PBLs  d e b r i s was  PBS  KC1  of  Sonic  of  3.0M  membranes;  sonication  Scientific  cell  180  cells  bursts,  Fisher  pelleted for  -10  sec  freezing  cell  METHODS  9  membranes  in  of  isolated  &  a  pool  immunized Freund's  of  normal  with.this complete  PBL  cell  antiserum, adjuvant  1 0  in  four  were  distal  immunized  schedule, in  intramuscular  bled  the ELISA  monthly  and>  weekly.  Activity  (24). This  procedure  to raise  antiserum  was  used  to raise  carcinoma antigens  was  reacted  order This  of antiserum  together  which  the p r e c i p i t a t e  to  bring  a further  containing anti-normal AML  cell  soluble  and  24  o f AML  ml  was  line  P815  ( 1 9 ) . The  human  o f AML  membranes i n  cell  f o r immune  membrane  precipitation. were  h,  by c e n t r i f u g a t i o n ,  added  antiserum  extract  a t 4 ° C f o r 24  following and more  to the s o l u b l e  The m i x t u r e  antigenic  used  precipitated i n 1.0  allowed  mixture  ml v o l u m e s  i n t r a p e r i t o n e a 1 1 y ) t o young  to stand  complexes  components  of  of the  the tumour-associa ted  t o immunize from  of  supernatent,  antibodies, soluble  and p o s s i b l y was  was  c e n t r i f u g e d . The  and normal  cells,  and a d m i n i s t e r e d 0.5  human  extract  m a t e r i a l was  bronchiogenic  c o n c e n t r a t i o n of 10-fold excess  h and then  antibody  method p r e v i o u s l y  Anti-normal  cell  removed  antiserum.  anti-normal  membrane  antigen(s)  alum  human  anti-AML  tumour-associated  cell  to stand  antiserum  i t to a f i n a l  anti-normal for  human  f o r a human  proportions  and a l l o w e d  estimated  inttheffeedback This  to  was  follows:  a n d AML  was  used  Rabbits  immunization  antiserum  the feedback  KC1 extract  optimal  mixed  anti-normal  was  mastocytoma o u t as  week  antiserum.  specific  a 3.0M  to determine ratio  6-8  (25) a n d a n t i s e r u m  carried  with  a  antiserum  antiserum  i n 250 u l v o l u m e s .  of this  utilizing  i n the murine was  after  an a n t i - A M L  produced  antigen  procedure  sites  rabbits.  the supernatent  The with  (0.5 ml i n t r a m u s c u l a r l y adult  female  albino  11  rabbits.  Rabbits  interval, The on  antiserum  prior  were  eluted a  50%  testing  used  to  ELISA,  was  found  to  prepared antiserum  with  normal  to  from  4-6  and  was  week  bled  weekly.  absorbed  normal  PBL  twice  membrane  ELISA.  4  This  was  1.0  sites.  later  and  membrane  following  and  on  by  were  rabbits. adjuvant  given  after  testing of  the  in  the  anti-normal  immunoadsorbent extracts.  hyperimmunizing  p r e p a r a t i o n and  several  the  injected  1 week  following  membrane  prepared  complete  levels  in  albino  Rabbits  adsorbed  cell  activity  ml  bled  insignificant  also  weekly,  of  preparative  female  Freund's  antiserum,  normal  cell  adult  volume  not  from  antigenic  with  distal  weeks  have  protein,  young  total  four  with  human  rabbits  rabbits  have  mixed  t h e r e f o r e was  normal  these  a  into  immunization.  a  was  to  second  columns  shown  immunization  and  i n the  a  immunized  prepared  immunize  material  emulsion  activity  these  after  ( a n t i - A M L # 1 ,#2,#3) :  gels,  intramuscularly another  from  bled  were  containing eluted  polyacrylamide  The  to  were  they  columns,  antiserum  Fractions  ELISA  which  recovered  immunoadsorbent  Anti-AML  immunized  following  extracts,  in  so  monthly  An  anti-  rabbits  bleeding immunizations.  Immunoadsorbents; Immunoadsorbents or  normal  cell  containing anti-normal  membrane  Antiserum  raised  membranes  or  extracts  in rabbits  antigen  to  a  human  were  prepared  pool  of  e x t r a c t e d from  these  normal same  antiserum  as  follows:  PBL normal  cell PBL  1 2  membranes  was  coupled  4B  (Pharmacia  to  use, the immunoadsorbent  borate  Chemicals,  buffered saline,  columns borate in  Fine  to cyanogen  and c y c l e d  over  buffered saline.  this  remove  manner  prior  pH  columns 8.5.  twice,  enriched  f o r tumour-associated  additional were  studies  antiserum.  cycled  over  This  and  columns  normal  cell  remove  the m a j o r i t y of the a n t i - n o r m a l  in  components.  these  anti-AML  antisera,  absorbed  anti-AML  antisera  ELISA each  a n d FACS  with  equilibration  with  Enzyme-linked  immunosorbent  sorbent  were  assay  protocol  normal  P B S , pH  assayed  (ELISA)  cell  gels  assay  using  eluted  or a n t i g e n  membrane  0.1N  presumably were  produce  preparations  was  immobilized found  activity  by t h e E L I S A .  for further  HC1  to  cell  components,  treatment  columns  treated  found  containing  human  using  i n the  to  the  were  present The  studies purged  to  i n the  after  f o l l o w e d by  7.2.  following  (2 ) . B r i e f l y ,  poyacrylamide  used  IV. Immunoadsorbent  a d s o r p t i o n by w a s h i n g  Antisera  This  as d e t e c t e d were  onto  fractions,  antiserum  Prior  with  were  was  present  immunizations  immunoadsorbent  membrane  samples  membrane  Some a n t i - A M L  layered  treatment  pass"  cell  (26,27).  a 90 m i n p e r i o d ,  membrane  "column  Sepharose  equilibrated were  90% o f t h e p r o t e i n  p r e p a r a t i o n s . The  f o r subsequent  were  during  activated  Sweden)  Samples  A l lc e l l  membrane  used  Uppsala,  to testing.  approximately  bromide  extracts  (ELISA);  the enzyme-linked a previously reactive  was  coated  described  material  i n t h e form  immuno-  from  of absorbed onto  ELISA  native AML  or  microtitre  13 plates  (Cooke  0.05M  carbonate  at  4°C.  buffer  Engineering,  Plates and  buffer, were  reacted  tempeature  and  20  Sheep  buffer.  coupled then  to  dilution. at  room  again  to  with  washed  the  ELISA was  phosphatase  activity  104)  min  for  absorbance  Laboratories, row at  of  wells  least The  with  AML  of  and was  band#1 for  100  allowed  to  proceed  buffer  with  room  and  band#1  in also  the  Plates  triplicate used  to  on  dilutions  with of  equivalent after  was  which  ELISA  p l a t e s which  min  (from  a l l preparations had  been  were  for  1/1500  90  min  washed  alkaline (Sigma read  (Flow  blanked  against  a l l tests  out  inhibition  were  at  AML  studies  405  for  with  the  so ELISA  temperature.  concentrations  ug/ml)  tested  in  were  membrane  selected nm  room  various  3.0  cell  were  a  run  ( a n t i - A M L # 1 , #2 , #3)  antisera  pre-coated  a  further  absorbed  to  at  occassion.  with  room  was  p l a t e s were  incubation at  0.0  (28),  Multiskan  and  at  PBS-Tween  phosphate-Na  absorbances  pre-incubated  material  after  45  each  VII)  a  h  glutaraldehyde  each  caary  the  min  with  volumes  were  alone  60  been  ELISA  Titer.tek  Ca..). i  20  p l a t e s were  p-nitrophenyl  in a  PBS-Tween  developed  temperature.  nm  ul  in  18  times  Type  material. A l l antisera  and  antiserum  AML  (Sigma  ng/ml for  for  had  in  reactivity  antigen  three  which  300  volumes  antiserum  Ig,  containing antigen  a l l yielded  ori-.this  test  at  with  plates  20  405  ul  times  additional  Inglewood,  ELISA  preparation  Each  at  twice  normalized  that  at  100  f o l l o w i n g which  PBS-Tween  45  three  the  an  in  phosphatase  temperature  for  9.6,  anti-rabbit  Reaction  with  pH  washed  alkaline  added  A l e x a n d r i a , Va.)  for  h  at  reactivity  on  absorbed  18  AML  4°C,  cell  14  membrane  extract  Polyacrylamide  at  gel  300  electrophoresis  Electrophoresis of  Laemmli  dodecyl  (29),  (SDS)  Samples  applied  to  7.5%  was  except  sulphate  omitted.  of  gels  weight  loading  with  albumin  (67,000  anhydrase and  or  silver gels  remainder  slicing  and  Elution  of  were  up cut  locations  ug  were  cell  a  being  gel s, f o r  of  a  onto  accomplished  were  extracts  were  to  were  to  of  the  gel  stored  at  -70°C  was  until  for (1.5  5-6 mm  gel.  bovine  h.  x  10.0  Molecular  serum  dalton),  of et  carbonic  (20,100  stained  Wray  and  co-electrophoresing  procedure  according  70V  comb  inhibitor  Gels the  at  by  (43,000  trypsin  analytical  the  containing  section  of  (2-ME)  one-tooth  protein  procedure  sodium  both  :  the  the  electrophoresed  dalton).  stained  to  instances  membrane  ovalbumin  according  according  2-mercaptoethanol  using  dalton),  elution  with  dalton) either  Fairbanks a 1. (31) .  removed i t was  et  al.  For  and  stained,  required  for  protein.  protein;  Stained lined  in  standards,  (14,400  Blue,  preparative the  run  dalton),  (30,000  Coomassie  some  and  250-500  protein  lysozyme  (30),  were  determinations  samples  that  polyacrylamide  Preparative and  out  absorbed  procedures,  (PAGE);  carried  and  preparative  cm)  ng/ml.  and  and from on  unstained  illuminated regions the  of  stained  portions from the  below  of  preparative  using  unstained  section  of  the  a  gel gel  light  gels  were  box.  Bands  corresponding shown  to  to  contain  15  detectable  protein.  glass  tubes  added  to  mixing  and  each  in  a  Gel  crushed  tube  and  Labquake  slices  were  with  spatula.  a  elution  rotator  was  placed  in  2.0  small,  capped  ml  of  by  end-over-end  effected  (Labindustries,  PBS  were  Berkeley,  Ca.)  o at  4  C  for  48  h.  Eluted  filtration  and  determined  using  the  material  eluted  by  using  a  protein  rabbit  Two-dimensional Gel  100  to  ul ul  of  2.25  ul  2-ME  (2%  final  8  ul  mg  of  a  solid  between the  at  tube  gel  short  segment  a  ml  of  1.0 the  tube  gel  was  al.  activity  antiserum  (23). was  (19,20)  band#1  material  was  were  Testing  of  accomplished  and  the  analysed  ELISA.  (2D-PAGE) ; on  2D-PAGE  et  al.  (35).  In  containing  36  ul  band#1  (40.0  ug/ml),  Tris-HCl final 5  (8M  was of  to then  40%  X-100  final  gel  and  of  (4%  AML  4.5 LKB  ul  the  of  Tygon  was  After  from  tubing  filled  with  the  prepared  a  (8%  the to  added  for  20%  to  2 mm  final  cone),  cone)  and  a  single  isoelectric  x  11.5  (V-h)  cm)  totalled  by  a  tube of  clean  running  the  water  in  at  glass the  55  and  coated top  cone),  solution  focussing  the  onto  SDS (2%  brief,  final  isoelectric  distilled  gel  (0.05M  final  "volt-hours"  10,000V-h. extruded  6.8  Ampholines  solution  cone.)  pH  polyacrylamide,  until  push  of  solution,  cone),  ul  Triton  syringe  tubing  antigenic  et  a l l fractions  O'Farrell  400V  7,500  Lowry  vacuum  of  cone),  tube  of  of  by  procedure  (5%  10%  for  anti-AML  1.0M  urea  focussing focussed  a  collected  concentrations  method  AML  the  sample  2.25  was  p o l y a c r y l a m i d e r.gel ^ e l e c t r o p h o r e s i s  purified  according a  the  protein  (IEF),  attaching  tube the  and  using  other  end  plate.  second  a  The  dimension  16  by  equilibrating  50mM 2  h  Tris-HCl with  of  the  into  After  position to  according  former  a  Junior  in  the  1.5  mm  1%  the  was  when  were  Orbit tube  of  3.0  Wray in  added  for  least  Shaker gel pH  run et  the  The  was  al.  at  was  room  set  6.8)  on  and  in  top  secured  running contact  completed, (31).  second  area.  stained  Molecular  dimension  using  a  well-former  solidified  and  before  running  the  at  previously described  overlay  cm).  had  under  the  agarose  overlay  2-ME  gel-stacking gel  run  included  ;x  the  5%  agarose -.solution  tube  then  1%  and  glycine,  by  plastic was  removed  molecular of  well-  weight  the  second  commenced.  human  R.C. 1640  the  medium  N.C.I.,  supplemented 37°C  cell  prior  containing described  promyelocytic  Gallo,  humidified when  6.8,  (384mM  line; The  RPMI  over  were  standards  dimansion  Dr.  x  pH  buffer  polyacrylamide,  a  procedure  agarose  protein  Cell  the  well  (5 mm  the  (3%  (cf. above),and to  a  on  allowing  standards  forming  when  by  bed  dimension  conditions  SDS),  running  equilibration,  gel  solidify  second  weight  0.1%  SDS-PAGE  agitation  stacking  buffer The  and  gentle  temperature.  i t with  to  cell  medium below.  and  line  HL-60  Md.  I t was  Bethesda, with  incubator  density  cell  10%  in  fetal  10%  reached membrane labelled  CO.,.  1 x  calf  10^/ml,  FACS  were  washed or  obtained  maintained  serum  Cells  preparation for  was  from in  in a harvested in  washed  serum-free in  IV a n a l y s i s , - a s  serum-  1 7  Fluorescent  antibodies;  Fluorescein-labelled was p r e p a r e d the  goat  after  according  antibody  which  D E A E - p u r i f i e d goat  to a standard  (10 mg/ml)  i t was  dialyzed  was  pH  8.5, a n d f i n a l l y  buffered  saline,  pH  9.2.  P B S , pH  isothiocyanate passage  (F:P)  of^r4 was  Cell  5%  serum,  1/10  were  over  from  Sephadex A ratio  bicarbonate  0.05M  bicarbonate  terminated  FITC-IgG G-25  by  dialysis  fluorescein  conjugates  (Pharmacia  of fluorochrome  by  Fine to protein  by s p e c t r o p h o t o m e t r i c a n a l y s i s ( 3 3 ) .  calf  anti-AML  i n P B S , pH  to eliminate  PBS  a n d 0.2  was  added  1.5  h on  and washed  twice  i n PBS  following  which  1 x  serum,  h on i c e w i t h  7.2. A l l a n t i s e r a Cells  ml o f f l u o r e s c e i n a t e d cell  i c e , washed calf  pellet.  once  serum.  0.2  or a n t i - n o r m a l  aggregates.  t o each  fetal  harvested  f o r 1.5  use  ml  removed  Sweden).  fetal  incubated  rabbit  100%  was  determined  cells  containing  to  was  NaCl,  labelling; HL-60  were  (FITC)  Uppsala^  The r e a c t i o n  0.15M  7.2, a t 4 ° C f o r 4 h . U n b o u n d  of the material  Chemicals,  0.5M  against  IgG  (32). B r i e f l y ,  against  for 5 h against  saline,  0.02M  procedure  dialyzed  buffered  against  anti-rabbit  Cells  calf  were serum  antiserum)  centrifuged  washed  three  anti-rabbit  were  i n PBS, a n d t h e n  The c e l l s  PBS c o n t a i n i n g 5% f e t a l  were  goat  cells  ml o f a n t i s e r u m  human  were  10  before times i n  a  centrifuged  f o r FACS  suspended IV  diluted  I g G a t 1/20  incubated  finally  (normal  further through i n 1.0  analysis.  18  Fluorescence-activated Twenty-five analyzed length power was  on  of  using  the  of  um  in  showed  positive  control  inhibition  Model  The  filter).  The  and  material  on  same  (anti-normal  for  18  antiserum  h  on  only  4°C  antisera,  were  extracts  the  HL-60  the  cells  and  antiserum antisera  using  Antisera  of  cell  the  as  the  as  a  was  result,  used.  (normal AML  were  inhibition absorbed  (d =  A l l anti-AML  diluted  These  and  HL-60  reactivity  antisera  well.  (34)  Pa.).  the  IV.  analysis  microspheres  with  repeated as  cells  absorbed  ug/ml.  a  standardized  reactivity  control  at  was  were  40.0  i n "..'the FACS  laser  wave-  FITC  antiserum.  same  nm  for  against  the  the  and  with  at  with  were  488  Warrington,  anti-AML#1  human)  antigen  HL-60  membrane  at  IV  blood  titrated  human)  approximately  anti-normal  red  Inc.,  compared  (anti-AML#1)  inhibiting  tested  and  the  filter FACS  sample  argon  carboxymethyla ted  were  analysis;  using  164-05  standard  Polysciences  studies,  antiserum  serum  cell  mW.  IV  antisera  the  Physics  the  control  the  IV  antisera  positive  test  FACS  anti-AML FACS  cell  Dickinson  pass  SD;  IV  each  monodispersed  0.02  (FACS)  from  glutaraldehyde-fixed chicken  All  in  400  sorter  cells  Spectra  (520-long  fluorescent  line  thousand  Becton  setting  used  1.75  a  cell  Both rabbit  band#1 1/10  and  subsequently  assays, normal  using  human  1 9  RESULTS  Antiserum feedback  normal  are  shown  human  cell  equivalent  that  this  These  antiserum  cells  which  least  in this  serum  was  that  cell  cell  a  blast  are also  lineage.  The  would  results  that  the antigen  i s not present  cell  population,  regardless  came  from  clinincally  hematopoetic In of  stem  subsequent  a pool  equivalent  cell  normal  of the  fact  to  AML  extracts, at that  this  the  blast  by  assumption  cells  i n Figure  i n detectable  anti-  differentiation  investigated on  anti-  of the  2 indicate  levels  in either  t h e bone marrow  i n d i v i d u a l s or those  with  sample various  disorders.  human  preparation  shown  o f whether  normal  studies,  of normal  contain  absorbed  common  or a  cells,  12  w h e r e a s no  cell  was  and  the anti-AML  indicative  antigen  AML results  16 AML  using  The p o s s i b i l i t y  o f bone marrow  population  of  an a n t i g e n  cells  both  a n y o f t h e 12  i n normal  cell  lymphoid  that  with  antibody  Representative  extracts  observed  results  on  extracts  myeloid  cell  procedure.  detecting  examining this  was  the  against  extracts  show  i s recognizing  assay  using  i n the ELISA  results  i s not d e t e c t a b l e  not found  KC1  assayed  a l l 16 AML  reactivity  extracts.  AML,  i n the ELISA  i n which  These  with  cell  tested  were  antiserum.  against  membrane-extracts.  1,  samples  reacted  antigen  and  i n Figure  cell  anti-AML serum  prepared  procedure,  and  normal  was  sonicated white  o f AML  cell  blood  blast  cell  cells  membrane  preparations  membranes were  used.  and an After  20 Figure  1:  ELISA  normal  peripheral  AML  antiserum.  AML  cell  Normal  individual  blood  extracts:  cell  Anti-AML  of  leucocytes  (PBL)  (O).  extracts:  antiserum  extracts  at  (A).  1:100  dilution.  of  either  with  AML  absorbed  or anti-  2  0.3 ANTIGEN  1.0 CONCENTRATION  3.0  ug^m!  1  22  Figure cell  2:  ELISA  samples  with  of extracts either  from  AML  anti-AML  and v a r i o u s  or anti-normal  bone human  marrow anti-  serum.  a)  Antigen with  b)  preparations  variety  bone  Normal  2.  Lymphoma  patient  the  marrow.  4.  bone  bone  Multiple  Lymphoma  Acute  Normal  cell  Anti-AML  extracts  stem  with  cell  malignant  patient  with  with  o f two  patients  disorders  as  with follows:  cell  infiltration  malignant  in  cell  malignant  cell  patient  with  malignant  marrow.  marrow.  were  used  a t 3.0  human  ug/ml.  antisera  d i1u t ion; antiserum:  Anti-normal  patients  marrow.  leukemia  i n t h e bone  and a n t i - n o r m a l  Anti-AML  from  marrow.  minimal  i n t h e bone  lymphocytic  bone  extracts  i n t h e bone  patient  infiltration 6.  extracts  marrow.  myeloma  infiltration 5.  cell  of hematopoetic  infiltration  All  marrow  1.  3.  cell  AML.  Individual a  from  human  ) • antiserum:  were  used  a t 1:100  cell  2.0,  i.ok  ooo a a a o a a o a • a a a • a a a a a o a o ooo ooo  O 0^  ooo  GOO ODD OOO a a a a o a  a a a a a a ooo ooo o o a a o a a • o o a o • • o • a • ooo a • a ooo  ODD  o a a ooo a a a ooo 0 o • a o • • o • ooo ooo  O • D OOO  -Ell. 2  AMI  0  3  BONE  4 MARROW'*  5  24 absorption column, a  of  these  cell  containing  anti-normal  polyacrylamide  7.5%  pattern  emerged  displayed  membrane  in  g e l ,  which  single  protein  preparation  showed  the  Preparative  gels  were  and  each  four  protein  for  reactivity  the  with  ELISA, (19).  This  be  whereas  reactive  above for gels  was  used  antiserum inrthe  raise  for  and  c e l l  extracts  individuals  lymphoproliferative AML in  antiserum the  FACS  IV  with  and  bone  showed,  myeloproliferative  this  antiserum  with (20). shared  This  bands#1  no  similarly, disease  membrane  react  in  in  from  only  fluoresced  used  preparative This  s p e c i f i c i t y myelo-  or  c e l l with  using  this  variety  of  in  the  -.  reactivity  equivalent  cells  '„ to  #3  was  disease  a  the.  and  with  work,  (Fig.  sample  rabbits.  with  band  examined  absolute  apparent  that  and  from  patients  cells  bands  material  eluted  Further  marrow  protein  significant  band#3  a  membrane  bands#1,#2  no  on  preparation  antiserum  antiserum  not  with  antiserum and#2,  indicated  common  AML  running  of  antipatients  patients  presence  of  (21).  anti-AML#3  AML  did  c e l l  eluted  anti-AML  from  and  conditions,  c e l l  essentially  disorders.  with  The  were  showed  extracts  disease  AML  material  showed  AML  unique  the  showed  specific  p r o l i f e r a t i v e from  4  immunoadsorbent  membrane  the  C o n s e q u e n t l y , -AML  (anti-AML#3)  ELISA  on  band#4  studies  to  of  feedback  AML  cell  bands  examination  background.  preliminary  the  run  an  antiserum,  and  presence  on  non-reducing  normal  band  3).  of  human  under  the  a  extracts  eluted  that  antigenic  was  AML  sites  also from  found the  bands#1,#2 and  that  to  same and  this  react  strongly  preparative #3,  at  antigenic  gel  least, identity  Figure  Analytical  3  extracts  of  normal  gel pattern  non-reducing  human  blast cells  PBL a n d  from  from  an  membrane AML  pa t i e n t .  lane  a:  Molecular  lane  b:  Normal  lane  c:  AML  Both  cell  prior Gel  human  blast  membrane  immunoadsorbent  weight  standards.  PBL membrane  cell  membrane  preparations  column  extract.  extract.  had been  containing  absorbed  anti-normal  twice  human  on a n  antibody,  t o PAGE.  stained  (1971).  i n Coomassie  Blue  according  to Fairbanks  et  a l .  67.000 • 43.000 • 30.000 • 20,100 •  27 might of  be r e f l e c t e d  the considerable  #3,  forms  band#1)  (AML  In would  order  preparations  were  prepared,  gels  appear  procedures since  membrane  from  normal  stem  cell  band.  to confirm were  that  probably  feature  cells  represent constituent  other  g e l band  AML  cell  protein from  AML  membrane bands  of c e l l  o f AML  membrane  band#1  o f AML  extracts band#1.  of cells  Membrane  AML, d o n o t p o s s e s s  i s slightly  This  may  lower  represent cell  anti-AML#1  i n molecular  an u n m o d i f i e d  membrane  antiserum,  which  may  sample  weight  form  preparations  with  hematopoetic  t h e APML/AMML  membrane  react,  this  protein  (acute p r o - /  contains  a  band  AML  band#1.  band#1  i n that  than  o f AML  patients  preparations  than  that  material, from  d i s o r d e r s , other  cell  These  observed  individuals  leukemia)  samples  (Fig.4).  from  t o note  with  of the preparative  aggregates  between  would  patients  b a n d s # 2 a n d #3,  artifacts  patterns  or those  that  the  might  a n d r u n on 7.5% SDS-PAGE  It i s interesting  APML/AMML  from  preparations  i s the presence  monomyelocytic  Because  bands#1,#2 and  AML  weight  similar  d i s o r d e r s , a panel  absorbed  t h e common AML  molecular  run,or i f analogous  and r e p r e s e n t e d  with  b a n d s # 2 and#3  whether  extracts  lymphoproliferative were  between  as w e l l .  considered.  when  in cell  originally,  AML  to determine  develop  seen  that  of the lowest  was  simi1iarities  cross-reactivity  the p o s s i b i l i t y  aggregated  be  in structural  i n the ELISA,  be i n d i c a t i v e  of  with  structural  similarities. In #3  order  represent  to test  further  aggregates  the contention  o f AML  band#1,  that  antisera  AML  bands#2 and  to a l l three  28  Figure from  4:  S D S - P A G E o f a number o f a b s o r b e d  a variety  lanes  a-d:  of c e l l  Absorbed cell  e  :  Absorbed  lane  f  :  Molecular  g-i:  Absorbed bone  stained  (1981).  using  sources.  membrane  extracts  membrane  extract  of four  individual  o f a n AMML  weight  standards.  membrane  extracts  marrow and a n o r m a l  samples,  Gel  extracts  AML  samples.  lane  lanes  membrane  cell  of a pre-CLL,  human  buffy  coat  sample,  an ALL cell  respectively.  the s i l v e r  staining  method  o f Wray  et  a l .  29  30 protein  bands  antiserum  was  membrane white  blood  do  raised  tested  samples  antisera and  were  i n the ELISA  membranes.  specifically  not react  with  with  i n v e s t i g a t e the p o s s i b i l i t y  to  similar,  as  i f not i d e n t i c a l ,  inhibition  assays  the i n h i b i t o r .  100%  inhibited  1.0  ug/ml).  antisera that and be  by  This  the three  uniquely  AML  to  establish  2D-PAGE  result  that  protein  i n Figure  supports  bands  was  performed from  representative be s e e n  component mean  band#1  a variety  gel pattern  that  AML  band#1  of approximately  p i o f 7.16.  is  probably  #1  material.  due  The  was d i r e c t e d  i n t h e AML Since  become more  band#1 that  samples AML  band#1  tested  from and  i n Figure  order  band#1 samples.  A  7 i n which i t  68,000 d a l t o n  molecular  to d i f f e r e n t i a l  individuals  In  of a single  or s t r e a k i n g  to  structure  i s composed  "tailing"  antigenic  component,  AML  membrane  and  i s found  important.  of p u r i f i e d cell  (about  a l l three  share  constituted a single  i s shown  band  material  determinants  the character  o f AML  i n AML  band#1  o f AML  antigenic  o n a number  order  6, a l l t h e a n t i s e r a w e r e  i n a l l preparations  AML  sample  In  a l l present AML  human  a l l the  membrane  the contention  identity.  that  antiserum  using  concentration  and i t s o r i g i n that  each  each  cell  of normal  show  cell  of  AML  preparation.  antigens,  t o t h e same  structural  band#1  preparations  a  human  myeloproliferative disease,  of  can  shown  t h e same  present  (Fig.5)  were p e r f o r m e d  are directed  possibly  with  As  absorbed  t h e AML  the normal  specificity  preparation  Results  to  #1,  using  and an e q u i v a l e n t  cell  react  i n r a b b i t s . The  seen  protein weight  with  i n Figure  g l y c o s y l a t i o n o f t h e AML  band  7  3 1  Figure  5:  absorbed  The of  Specificity AML  antisera either  extracts  were  absorbed  of various i n the  titrated AML  anti-AML  antisera  for  ELISA.  on  ELISA  antigen  wells  or absorbed  c o n t a i n i n g 300 normal  human  antigen;  a n t i -AML #1  antiserum  II  a n t i -AML# 2 a n t i s e r u m II  a n t i -AML#3 II  antiserum  (on  AML  antigen)  (on  normal  (on  AML  (on  normal  (on  AML  (on  normal  ( •  antigen)  antigen)  (  antigen)  antigen)  (  antigen)  ) • (A  •  )  ) -  (O ) • ) •  ( • )  ng/ml  32  -LOG  OF  DILUTION  33 Figure  6:  various  Anti-AML  Inhibition  concentrations  antisera  concentrations  of  AML  AML  antigen  ng/ml  the  absorbed  anti-AML  of  which  absorbed of  of  AML  had  in  the  AML  ELISA.  antigen.  antiserum  ( A  )•  anti-AML#2  antiserum  (  •  ).  anti-AML#3  antiserum  (  •  ).  are  presented  as  percent  the  pre-incubated  were  anti-AML#1  Data  in  ELISA  with  band#1.  been  band#1  antisera  tested ELISA The  for  various  reactivity  wells  antisera  inhibition.  with  contained are  as  on 300 follows;  -2.5 -1.5 -0.5 0.0 0.5 (0.003 0.03 0.3 1.0 3.0 LOG CONC. INHIBITOR  ug/mi)  35  Figure  7:  Two-dimensional  polyacrylamide  gel profile  of  AML  loaded  an I E F  band* 1 .  lane  a:  Molecular  lane  b:  5 ug o f AML tube was  These any  IEF tube  was Wray  seen  then  silver  et a l .  lines  present)  t h e SDS-PAGE  SDS-PAGE  placed  sample i t was  running  onto  a  7.5%  a t 70V f o r 5 h . T h e  PA  finished  t o t h e method  (1981).  are artifacts appear  The  which  stained according  on t h e g e l between  weight  with  g e l was  gel  same  procedure.  a t 400V f o r 24h, a f t e r  and e q u i l i b r a t e d The  onto  2% LKB A m p h o l i n e s .  and e l e c t r o p h o r e s e d  protein  either  m a t e r i a l was  gel  of  molecular  band#1  focussed  buffer.  lines  standards.  gel containing  extruded  The  weight  on  o f t h e SDS-PAGE  'blank  and t h e r e f o r e running  67,000 and 55,000  gels'  (gels  represent  dalton  procedure. run  without  artifacts  c o n d i t i o n s or the  of  ,  si1ver-staining  36  37  Earlier cells a  studies  i n t h e FACS  cell  surface  component  with  HL-60  manner the  with  indeed  AML  carried  to react  t o the anti-AML#3  with  which  band#1, an  anti-AML#1  inhibition  o u t . I t c a n be s e e n  blocks  the r e a c t i o n  (Fig.8a  and T a b l e  I ) . Conversely,  successfully  normal  antiserum  human  partial AML by  band#1  cell  components.  of both  The p r e s e n c e  moieties  together  a  of  result  on  antiserum normal  of both  molecule  most  cells  membrane  of the  anti-  I ) . The  antiserum  by  antiserum  ( F i g . 9 a and  the p o s s i b i l i t y  'leukemic'  was  successfully  HL-60  cell  human  preparation  'leukemic-transformation'  c e l l (s ).  material with  that  I V was  and T a b l e  leukemia-associated  in a  HL-60  I) a n d t h e a n t i - A M L # 1  f o r by  t h e same  on  human  (Fig.9b  reacted  To e s t a b l i s h  i n t h e FACS  band#1  with  reacted  cells  reacting  the anti-normal  membrane  I) c a n be a c c o u n t e d  also  i t also  the r e a c t i v i t y  HL-60  and T a b l e  human  i s comprised  stem  with  of both  (Fig.8b  the normal  Table #1  inhibition  block  was  AML  of anti-AML#1  preparations  antiserum.  detected  i n t h e FACS I V .  patients'  assay  that  HL-60  showed t h a t  patients'  of p a t i e n t s  antiserum  line  with  on  antiserum  on c e l l s  (20). This cell  antiserum  this  only  antiserum  and appeared  material  that  present  leukemia  anti-AML#1  identical  shown  disorders  the promyelocytic  Studies  the anti-AML#3  IV have  myeloproliferative with  using  that  AML  and normal and  of normal  cellular  'normal'  probably  band  cellular  arises  hematopoetic  as  38  Table  1.  Results  antisera human  o f FACS  pre-incubated  cell  membrane  IV a n a l y s i s  with  AML  antigen  o f HL-60  band#1,  o r PBS  prior  cells  absorbed  with  normal  to l a b e l l i n g  and  analysis.  Number Antiserum  Normal  rabbit  Fluorescing  -  3787  antigen  4127  normal AML  Anti-normal "  band#1  human  normal AML  22,392 3560 20,927  -  13,175  antigen  13,370  normal AML  antigen  34 8 5  band#1  A n t i - A M L #1 "  Cells  Inhibitor  serum  "  of  band#1  4547  39  Figure  8:  Inhibition  human  antisera  panel  a:  with  AML  Inhibition the  FACS  pattern  panel  b:  shows  HL-60 The of  o f AML  cells  upper HL-60  The with  lower  with  AML  cells.  band#1  material in  The u p p e r  with  distribution cells  distribution  anti-AML#1  that  to i n h i b i t  with pattern  has been p r e -  the fluorescence of  anti-normal  pattern  human  pattern  antiserum.  the f l u o r e s e n c e human  shows  antiserum  band#1.  human  shows  the anti-normal  distribution  AML  The l o w e r  with  distribution  with  by AML  band#1.  band#1  reacted  cells  anti-normal  t h e f l u o r e s c e n c e o f HL-60  anti-normal  incubated  HL-60  cells  with  and  i n t h e FACS.  antiserum.  HL-60  Failure  band#1  IV u s i n g  incubated  o f anti-AML#1  o f anti-AML#1  anti-AML#1 shows  analysis  that  HL-60  antiserum. cells  has been p r e -  4 1  Figure human in  9:  Inhibition  antisera  with  analysis  absorbed  o f anti-AML#1  normal  human  and  cell  anti-normal  membrane  antigen  t h e FACS.  panel  a:  Failure  of absorbed  antigen  to i n h i b i t  reacted  with  fluorescence anti-AML#1 antigen.  the panel  b:,  pattern  antiserum  indicates  fluorescence  of normal  of anti-normal  human  cell  membrane  HL-60  cells.  The u p p e r  the absence  lower  line  presence  line  of normal  demonstrates  of the normal  upper of the  of normal  pattern with  human  shows t h e HL-60 i n  antiserum  i n t h e FACS indicates  human  cell  by IV  normal  using  fluoresence  antigen,  the i n h i b i t i o n human  cells  antigen.  human  antigen  o f HL-60  reactivity  antiserum  human  membrane  The  i n the absence  Inhibition  in  cell  antiserum.  o f anti-AML#1  presence  human  the fluorescence  anti-AML#1  The l o w e r  reactivity  normal  whereas the seen  membrane  i n the antigen.  43  DISCUSSION  The 20), of  data  show  presented  that  the a n t i s e r a  gel purified  cells  react  with  and  from  antisera  marrow  disorders  cells  cells  from  that  In a p r e v i o u s  membrane  preparations  demonstrated  logically  PAGE,  normal  t o some  w a y s . When  only  membrane  present.  preparations  myeloproliferative identical  cell  this  studies,  myeloproliferative IV  d i d not  with  a  (21),  that  cell  when  r u n on  of these membranes band  cells  the  cell  absorbed  u n i q u e , immunotime,  we  proteins  have in  a r e r u n on  (AML  present  SDS-  band!1) i s in a l l  of patients  and has n o t y e t been of c e l l s  .....  non-reducing  At t h i s  has been  preparations  whereas  showed  of 3 apparently  from  i n which  ( 2 0 ) , we  This  examined  of  on  weight  band  cell  present  molecular  membrane  of  specificity  with  bands.  AML  membrane  was  cells,  AML  disorders  cell  i n t h e FACS  the nature  absorbed  AML  of  those  disorders  protein  injection  extracts  i n d i v i d u a l s or those  the presence  the lowest  consistently  patients  i n question  o f AML  by  (19,  i n d i v i d u a l s or  i n further  publication  extent,  with  The  the a n t i s e r a  cross-reactive  clarified, various  from  the antigen  surface.  membrane  normal  confirmed  previously  preparations  disorders.  of l y m p h o p r o l i f e r a t i v e  indicating  PAGE,  absorbed  clinically  with  published in rabbits  equivalent  a n d PBL  reacted  comparable variety  was  that  i n the ELISA,  not with  either  and  prepared  from  lymphoproliferative  anti-AML bone  protein  exclusively,  preparations membranes  here,  from  cell  with  detected normal  in  44  individuals  or  those  disorders.  Antisera  present  the  in  the  in  ELISA,  confirm bands  the  seen  aggregates cellular seen  with  contention some  of  AML  in Figure  band#1, this  seen  in  and  of  a  pi  weight  of  used  in  3  Figures At  to  and  present,  antigen  present  cell  membrane  on  the  protein 240,000  weight  2D-PAGE,  of  the  68,000  discrepancy  in  the  AML  cell  daltons AML and  protein  of  the  bands  daltons  weight  absorbed  nature  a  percentage  of  high  finding  that  the  AML  patients  percentage  of  CML  daltons  molecular  different  membrane  the  noramally  patients that  both  antigen with  and  bone  Most  the  surface  p l e u r i p o t e n t stem  of  marrow  PAGE  sample  active  disease  present  differentiating  cell  but  i s an  this  to  band be  and  has  PBL  been  on  the  also  lymphoid  remission. of  AML  cells  interesting, only  expression  the  shown  i s not  in clinical  imply  origin  r e c e n t l y been  (21).  observations a  after  other  4  and  protein  represent  68,000  to  precise  or  of  110,000  due  has  of  the  likely  It  cells  of  data  4.  AML  myeloid  the  weight  some  molecular  molecular  i s most  with  high  since  known.  of  The  inhibitable,  m a t e r i a l . These  preparations  weights  bands  are  molecular  i s approximately  i s not  cells  sample  band#1  80,000,  apparent  run  protein  i n a s s o c i a t i o n with  4,  patients  blast a  higher  56,000,  The  band#1  conditions  the  the  apparent  7.16.  3  membrane  molecular  Figure  lymphoproliferative  the  that  respectively.  an  AML  cell  of  AML  i s further confirmed with  of  The  are  weight  migrates  #1  AML  each  purified  band#1  3  to  AML  gel  in  p a t i e n t s with  raised  absorbed  moieties.  molecular  from  on and  These  antigen  indication  of  on a  the  45  potentially have on  malignant  n o t been  cells  from  leukemia  able  change  to demonstrate  normal  individuals  o r lymphomas.  regarding  the o r i g i n  There  of this  gene  product  since  cell  surface  and a r e u s u a l l y  possible antigen such  a  normal are  that which  these  i s present  presently  addressing various  under  copy  number  not  the antigen  is a  antigen  and a d d r e s s  gene(s)  product  dominance bone  o f AML  outcome  of these  appears  t o be  expression  (blast  the question  or  i n AML  and  gene  CML  represent  at the  It i s also  and on  detected  These  in  possibilities  studies  product.  are also antigen  cells,  on  etc.),  this  putative of  of leukemia  stem  the importance  At p r e s e n t ,  expression  patients.  cells.  viral  f u n c t i o n and whether  o f how  due  a  differentiation  remission  encoding  malignant  clinical  lymphocytic  o f t h e AML  to the onset  remission  studies,  largely  Ongoing  i t spossible  antigen  t o be p r e s e n t  here.  cells,  the gene(s)  of a p o t e n t i a l l y  marrow  used  we  possibilities  normal  and d i s t r i b u t i o n  i s related  with  i t has not been  recombinant  i s to clone  of t h i s  glycosylated. a  time,  low c o n c e n t r a t i o n s  the tests  per c e l l ,  this  of  I t may  shown  highly  a t such  populations  the  been  investigation.  t h e number  cell  intention  by  number  antigen.  that  At  or p a t i e n t s  represents  of c e l l s  preparations,  cell.  the presence  are a  have  the antigen  l o w number  i n that  cell  the AML  this  or to the  clone  i n the  Regardless  of the  of t h i s  AML  to i t s apparent  or  antigen specific  46  REFERENCES  Cooper, Nature  G.M., O k e n q u i s t , 284, 418-421.  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