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Cytogenetic analysis of single hemopoietic colonies Dubé, Ian David 1980

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CYTOGENETIC ANALYSIS OF SINGLE HEMOPOIETIC COLONIES  by  ,  %  IAN DAVID DUBE •Sc., The University o f B r i t i s h Columbia, 1977  A THESIS SUBMITTED IN. PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES Department of Medical Genetics  We accept t h i s thesis as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1980  Ian David Dube, 1980  In  presenting  this  thesis in partial  an advanced d e g r e e a t the I  Library  further  for  shall  agree  the U n i v e r s i t y  make  that  it  this  written  thesis for  financial gain shall  Medical  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  by t h e  i s understood  permission.  Department nf  September 10, 1980  Genetics Columbia  I agree  a v a i l a b l e for r e f e r e n c e a n d  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 It  of t h e r e q u i r e m e n t s  of B r i t i s h C o l u m b i a ,  p e r m i s s i o n for e x t e n s i v e  by h i s r e p r e s e n t a t i v e s . of  freely  fulfilment  c o p y i n g of t h i s  not  copying or  that  study. thesis  Head o f my D e p a r t m e n t  that  for  or  publication  be a l l o w e d w i t h o u t  my  -ii-  ABSTRACT  C y t o g e n e t i c a n a l y s i s o f f r e s h marrow p r o v i d e s an p r i m a r i l y o f the d i s t r i b u t i o n of karyotypes compartments o f p r o l i f e r a t i n g h e m o p o i e t i c  assessment  i n the most d i f f e r e n t i a t e d  cells.  In t h e o r y ,  i t should  be p o s s i b l e t o o b t a i n s i m i l a r i n f o r m a t i o n f o r more p r i m i t i v e c e l l s i n c e , under a p p r o p r i a t e c o n d i t i o n s i n vitvo, form c o l o n i e s o f i d e n t i f i a b l e progeny. an approach, p a r t i c u l a r l y  types  t h e s e are s t i m u l a t e d t o  Although  the importance o f  i n s t u d i e s o f the hemopoietic  such  malignancies,  has been r e c o g n i z e d f o r a number o f y e a r s , c o l o n i e s o f more than 1 , 0 0 0 c e l l s a r e r a r e l y o b t a i n e d , and the a c q u i s i t i o n o f r e l i a b l e  severe t e c h n i c a l problems have hampered  data.  In t h i s work, a m i c r o t e c h n i q u e  has been developed  which  h i g h q u a l i t y chromosomes ( s u i t a b l e f o r G- o r Q-banding) t o be  enables obtained  from s i n g l e e r y t h r o i d c o l o n i e s grown from human b l o o d o r bone marrow i n standard m e t h y l c e l l u l o s e c u l t u r e s .  Thus, i t i s now  p o s s i b l e to begin  an  a n a l y s i s o f chromosomal changes i n t h e progeny o f i n d i v i d u a l e r y t h r o i d stem c e l l s .  T h i s p r o v i d e s a new  approach t o the c y t o g e n e t i c study o f the  o r i g i n and p r o g r e s s i o n o f the human m y e l o p r o l i f e r a t i v e d i s e a s e s .  -iii-  TABLE OF CONTENTS Page ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES  v i i  ACKNOWLEDGEMENTS  ix  INTRODUCTION 1.  2.  3.  4.  Chromosomal Involvement  i n Cancer  1  a.  Chromosomally Normal I n d i v i d u a l s — Marker Chromosomes and K a r y o t y p e E v o l u t i o n as F e a t u r e s o f M a l i g n a n c y  2  b.  Chromosomally Normal I n d i v i d u a l s — A s s o c i a t i o n o f S p e c i f i c Chromosome A b n o r m a l i t i e s With S p e c i f i c Malignancies  4  c.  Chromosomally  6  d.  P r a c t i c a l Implications  Abnormal  Individuals  8  The M y e l o p r o l i f e r a t i v e D i s o r d e r s  9  a.  Time Course  9  b.  Single .Cell O r i g i n  10  c.  Chromosome S t u d i e s  11  d.  The MPD as M a l i g n a n t C o n d i t i o n s  11 ;  The H e m o p o i e t i c System  12  a.  C o l o n y A s s a y s F o r P r i m i t i v e C e l l Types  12  b.  In Vitro  15  A s s a y s F o r Hemopoietic P r o g e n i t o r s  A p p l i c a t i o n o f Chromosomal A n a l y s i s t o S i n g l e Hemopoietic Colonies  17  a.  C h r o n i c M y e l o i d Leukemia  17  b.  Polycythemia Vera  c.  Present Objective  (PV)  (CML)  18 19  -iv-  TABLE OF CONTENTS Page MATERIALS AND  METHODS  1.  P a t i e n t Specimens  21  2.  Specimen P r e p a r a t i o n  21  3.  Chromosome H a r v e s t  25  4.  Chromosome Banding  28  5.  D i f f e r e n t i a l S t a i n i n g o f S i s t e r Chromatids  28  a.  Rationale  28  b.  Technique  29  i. ii. iii.  Bone Marrow  29  E r y t h r o p o i e t i c Colonies  30  Differential Staining  30  RESULTS 1.  Time Course a.  Cell  b.  Mitotic i. ii.  2.  32  Studies  32  Recovery  35  Index  Pooled  Colonies 37  Single Colonies  S i s t e r C h r o m a t i d Exchange  Studies  40  a.  F r e s h Marrow  41  b.  Erythroid Colonies  41  3.  Proof of the S i n g l e C e l l O r i g i n of I n d i v i d u a l l y Plucked C o l o n i e s  47  4.  K a r y o t y p e s From S i n g l e C o l o n i e s  49  TABLE OF CONTENTS  DISCUSSION  CONCLUSION  REFERENCES  APPENDIX 1A Procedure f o r the P r e p a r a t i o n o f Hemopoietic f o r P l a t i n g From F r e s h Bone Marrow A s p i r a t e  Stem C e l l s  APPENDIX IB Procedure f o r t h e P r e p a r a t i o n o f Hemopoietic f o r P l a t i n g From P e r i p h e r a l B l o o d  Stem C e l l s  APPENDIX IC Procedure f o r the P r e p a r a t i o n o f the C u l t u r e f o r Hemopoietic Stem C e l l s  Medium  -vi-  LIST OF  TABLES Page  Table I.  S p e c i f i c S t i m u l a t i n g Factors f o r Myeloid C e l l Lineages.  16  Table I I .  P a t i e n t Data.  Table I I I .  P e r c e n t a g e o f Recovered C e l l s i n Metaphase i n P o o l e d E r y t h r o i d C o l o n i e s H a r v e s t e d on D i f f e r e n t Days o f C u l t u r e . S i x C o l o n i e s Were P o o l e d t o G i v e Each V a l u e .  36  T a b l e IV.  Average Number o f S i s t e r Chromatid Exchanges Per C e l l i n P o o l e d E r y t h r o i d C o l o n i e s From Four I n d i v i d u a l s .  46  T a b l e V.  D i s t r i b u t i o n o f F l u o r e s c e n t Y-Body i n I n d i v i d u a l C o l o n i e s P l u c k e d From D i s h e s i n Which E q u a l Numbers o f Male and Female C e l l s Were P l a t e d .  48  Table VI.  Average Number o f A n a l y z a b l e Metaphases P e r Colony f o r S i x I n d i v i d u a l s . Ten C o l o n i e s Were P l u c k e d i n Each Case and Q- o r G-banded.  50  22-24  -vii-  LIST OF FIGURES Page F i g u r e 1.  The h e m o p o i e t i c system.. A schematic i l l u s t r a t i o n o f stem c e l l d i f f e r e n t i a t i o n showing lymphoid and m y e l o i d development from a common p l u r i p o t e n t stem c e l l .  13  F i g u r e 2.  Colony p l u c k i n g . Removal o f a s i n g l e h e m o p o i e t i c c o l o n y ( e s t i m a t e d s i z e 1,500 c e l l s ) u s i n g a f i n e l y drawn out P a s t e u r p i p e t t e .  27  F i g u r e 3.  T o t a l r e c o v e r y o f c e l l s from a s i n g l e c o l o n y a f t e r h y p o t o n i c t r e a t m e n t , f i x a t i o n , and two washes, shown as a f u n c t i o n o f t h e day o f c o l o n y h a r v e s t .  33/34  F i g u r e 4.  (a)  Cultures i n i t i a t e d c e l l s from ID.  from p e r i p h e r a l b l o o d  33  (b)  Cultures i n i t i a t e d c e l l s from KH.  from p e r i p h e r a l b l o o d  34  P e r c e n t a g e o f r e c o v e r e d c e l l s i n metaphase. Ten c o l o n i e s were p l u c k e d on each day f o r Days 9 t o 13 o f i n c u b a t i o n . A l s o shown a r e the a b s o l u t e number o f metaphases o b s e r v e d p e r c o l o n y .  38/39  (a)  Cultures i n i t i a t e d from A J .  from bone marrow c e l l s  38  (b)  Cultures i n i t i a t e d from HS.  from bone marrow c e l l s  39  F i g u r e 5.  C e l l c y c l e k i n e t i c s e v a l u a t i o n u s i n g 5BrdU i n c o r p o r a t i o n and t h e d i f f e r e n t i a l s t a i n i n g technique. Metaphases a f t e r (a) one, (b) two, and (c) t h r e e s y n t h e s e s i n 5BrdU. A l l t h r e e metaphases were t a k e n from one bone marrow c u l t u r e from an i n d i v i d u a l w i t h Bloom syndrome.  42/43  F i g u r e 6.  The number o f c e l l s i n f i r s t , second, and t h i r d metaphases a f t e r v a r i o u s l e n g t h s o f time i n i n c u b a t i o n i n 50 UM 5BrdU.  44/45  (a)  Cultures i n i t i a t e d from NF.  from bone marrow c e l l s  44  (b)  Cultures i n i t i a t e d from HS.  from bone marrow c e l l s  45  -viii-  LIST OF FIGURES Page F i g u r e 7.  Q-banded male k a r y o t y p e o b t a i n e d from a s i n g l e e r y t h r o i d c o l o n y grown from p e r i p h e r a l b l o o d c e l l s from WH.  51  F i g u r e 8.  Q-banded female k a r y o t y p e o b t a i n e d from a s i n g l e e r y t h r o i d c o l o n y grown from bone marrow c e l l s from EM.  52  F i g u r e 9.  G-banded male k a r y o t y p e o b t a i n e d from a s i n g l e e r y t h r o i d c o l o n y grown from bone marrow c e l l s from WS.  53  F i g u r e 10.  G-banded female k a r y o t y p e o b t a i n e d from a s i n g l e e r y t h r o i d c o l o n y grown from p e r i p h e r a l b l o o d c e l l s from EM.  54  -ix-  ACKNOWLEDGEMENTS  I would l i k e t o express my s i n c e r e . a p p r e c i a t i o n t o t h e chairman o f my t h e s i s committee, Dr. Connie Eaves ( M e d i c a l G e n e t i c s ) , f o r her c o n s t a n t s u p p o r t and guidance d u r i n g t h e p a s t y e a r . I would l i k e t o thank t h e o t h e r members o f my t h e s i s committee, Dr. F r e d D i l l (Medical G e n e t i c s ) , Dr. P a t r i c i a B a i r d (Medical G e n e t i c s , Departmental Head), Dr. Tom G r i g l i a t t i ( Z o o l o g y ) , and Dr. Tony G r i f f i t h s (Botany), f o r t h e i r guidance w i t h t h i s work. I would a l s o l i k e t o thank Dr. James M i l l e r ( M e d i c a l G e n e t i c s ) f o r h i s c r i t i c a l r e a d i n g o f t h i s m a n u s c r i p t and f o r h i s h e l p w i t h t h e o r g a n i z a t i o n o f my t h e s i s committees. I would espec i a l l y l i k e t o thank Dr. F r e d D i l l f o r p r o v i d i n g me w i t h t h e c y t o g e n e t i c t r a i n i n g t h a t made t h i s work p o s s i b l e , and Dr. K e i t h Humphries f o r our stimulating discussions. I w i s h t o thank t h e members o f t h e B r i t i s h Columbia Cancer Research C e n t r e f o r p r o v i d i n g an environment i n which i t was t r u l y a p l e a s u r e t o work. I am e s p e c i a l l y g r a t e f u l t o Dr.. A l l e n Eaves f o r h i s h e l p i n o b t a i n i n g t h e p a t i e n t m a t e r i a l s , and t o Dianne R e i d , Cam Smith, and Ann M a r i e MacDougall, f o r t h e i r e x p e r t t e c h n i c a l a s s i s t a n c e . I would a l s o l i k e t o thank Sharon Masui and t h e s t a f f a t t h e C y t o g e n e t i c s L a b o r a t o r y a t t h e Vancouver G e n e r a l H o s p i t a l f o r t h e i r h e l p f u l s u g g e s t i o n s , and Sharon B e l l f o r h e r p a t i e n t and e x p e r t a s s i s t a n c e . i n the preparation o f t h i s manuscript.  INTRODUCTION  1.  Chromosomal Involvement  i n Cancer  A t t e n t i o n was f i r s t drawn t o t h e s i g n i f i c a n c e o f chromosomal involvement  i n the transformation o f c e l l s  s t a t e i n 1914 by Theodor B o v e r i ( 1 ) . changes i n malignancy during the l a s t  remains  from a normal  t o a malignant  Today, t h e r o l e o f chromosomal  unclear.  Advances i n human c y t o g e n e t i c s  20 y e a r s have n o t r e s o l v e d t h e i s s u e .  We now know t h a t t h e g r e a t m a j o r i t y o f human c a n c e r s  exhibit  v i s i b l e chromosome changes arid t h a t e v e r y chromosome i n t h e human complement has been d e s c r i b e d as b e i n g i n v o l v e d i n one o r a n o t h e r t y p e o f k a r y o t y p i c anomaly  ( t r a n s l o c a t i o n , d e l e t i o n , d u p l i c a t i o n , t r i s o m y , monosomy,  e t c . ) i n human c a n c e r  ( f o r a review,  see Ref. 2 ) .  Some chromosomal  changes a r e i n v o l v e d more f r e q u e n t l y t h a n o t h e r s b u t t h e p r e c i s e s h i p o f t h e s e changes t o t h e development o f m a l i g n a n c y  relation-  i s n o t known.  In  p r a c t i c e , i t has u s u a l l y been i m p o s s i b l e t o o b t a i n chromosome p r e p a r a t i o n s from many tumor samples.  In a d d i t i o n , the q u a l i t y o f those p r e p a r a t i o n s  o b t a i n e d has o f t e n been poor because the chromosomes i n many c a s e s .  o f t h e condensed  and f u z z y n a t u r e o f  I n view o f t h e s e t e c h n i c a l problems,  i t is  not s u r p r i s i n g t h a t t h e a p p l i c a t i o n o f c y t o g e n e t i c f i n d i n g s t o t h e d i a g n o s i s and t h e r a p y o f human c a n c e r s has n o t been f u l l y e x p l o r e d . There a r e a few human c a n c e r s w i t h a c l e a r l y demonstrated and c o n s i s t e n t chromosomal involvement.  I n v e s t i g a t o r s have emphasized  these  i n t h e i r attempts  t o answer a key q u e s t i o n about t h e r o l e o f chromosomes  i n human c a n c e r :  i . e . whether chromosomal changes a r e c a u s a l o r conse-  quential to malignant transformation.  -1-  -2-  a.  Chromosomally Normal I n d i v i d u a l s — E v o l u t i o n as F e a t u r e s o f  Marker Chromosomes and  Karyotype  Malignancy  Most human tumors show d e v i a t i o n s from the normal d i p l o i d o f chromosomes (2). the presence  In g e n e r a l , two  striking  o f marker chromosomes, and  number  f e a t u r e s o f tumor c e l l s  a tendency  towards a  are  specific  p a t t e r n of karyotype e v o l u t i o n . Chromosomal markers a r e t h e r e s u l t o f rearrangements, r i s e t o new  chromosomes.  which g i v e  In some c a s e s , the marker chromosome can  i d e n t i f i e d i n terms o f the chromosomes t h a t g i v e r i s e t o i t , o t h e r s t h i s i s not p o s s i b l e .  but i n  Markers a r e v e r y commonly p r e s e n t i n tumors  w i t h h y p o d i p l o i d chromosome numbers. d i p l o i d and d i p l o i d tumors  be  (2).  T h e i r frequency  In one  i s lower  i n hyper-  study, markers were p r e s e n t i n  each o f 11 h y p o d i p l o i d o v a r i a n carcinomas,  and  i n 18 out o f 21 w i t h modal  numbers o f 46 o r more; t h e y were p r e s e n t i n each o f 12 h y p o d i p l o i d c e r v i c a l carcinomas  but i n o n l y e i g h t o f 18 tumors w i t h modes o f 46  or  more ( 3 ) .  In a more r e c e n t r e p o r t , k a r y o t y p i c a n a l y s i s o f 189  cases o f  carcinomas  o f the b l a d d e r r e v e a l e d t h a t t h e r e were marker chromosomes  i n 65 out o f 85 p r e p a r a t i o n s t h a t y i e l d e d a n a l y z a b l e metaphases ( 4 ) . Karyotype  e v o l u t i o n , a s t e p w i s e rearrangement o f the  o c c u r r i n g i n an a p p a r e n t l y o r d e r e d karyotypes.  Each i n d i v i d u a l tumor appears  d i r e c t i o n and e x t e n t o f any may  f a s h i o n , i s a second  f e a t u r e o f tumor  to vary with respect to the  change i n k a r y o t y p e .  be c l e a r when the average  karyotype  The  tendency,  however,  e x t e n t t o which chromosomes o f each  type  a r e under- o r o v e r r e p r e s e n t e d i n a s e r i e s o f tumors i s c a l c u l a t e d  (2).  An a n a l y s i s o f the "chromosomal d a t a o b t a i n e d i n a s e r i e s o f p a t i e n t s with chronic myeloid  leukemia  (CML), who  a l s o expressed  a s p e c i f i c marker,  the P h i l a d e l p h i a chromosome o f CML  (Ph" ) , suggested t h a t t h e c l i n i c a l  progression  1-  i s u s u a l l y accompanied by c o n c o m i t a n t p r o g r e s s i o n o f the  t y p i c p i c t u r e , and t h a t the l a t t e r may the d i s e a s e  (5).  karyo-  r e f l e c t the p r o g n o s t i c aspects o f  The P h ^ - p o s i t i v e c o n d i t i o n i n which t h e Ph^ i s t h e o n l y  k a r y o t y p i c a b e r r a t i o n appears t o be an e a r l y m a n i f e s t a t i o n o f  CML.  C l i n i c a l p r o g r e s s i o n o f t h e c h r o n i c phase o f P h ^ - p o s i t i v e CML  i s accom-  panied  by g r a d u a l p r o g r e s s i o n o f the c y t o g e n e t i c p i c t u r e , e s p e c i a l l y i n  t h e more t e r m i n a l b l a s t i c  stage o f the d i s e a s e  (6,7).  Meningiomas p r o v i d e a group o f tumors i n w h i c h t h e phenomenon o f k a r y o t y p i c e v o l u t i o n has been r e l a t i v e l y w e l l s t u d i e d . the c y t o g e n e t i c  f i n d i n g s i n o v e r 200 meningiomas,  t h a t the i n i t i a l  In h i s summary o f  Sandberg  (2) suggests  s t e p i s u s u a l l y the l o s s o r d e l e t i o n o f a #22,  and the  subsequent s t e p s a f f e c t chromosomes #8 and #9 i n group C ( i . e . #6 t o and #14  and #15  i n group D  A p o s s i b l e explanation  ( i . e . #13 t o #15),  #1.  f o r the e v o l u t i o n o f k a r y o t y p i c changes i n menin-  gioma c o u l d be the f o l l o w i n g : normal)  i n p a r t i c u l a r , and a l s o  #12)  Normal d i p l o i d  stem l i n e  ( a l l karyotypes  appearance o f a new v a r i a n t stem l i n e w i t h a growth advantage  :and showing monosomy f o r chromosome #22 ;and k a r y o t y p e s  (mixture  o f normal  karyotypes  w i t h monosomy 22 ->- i n c r e a s e i n t h e r e l a t i v e p r o p o r t i o n o f  c e l l s w i t h monosomy 22) -*- subsequent k a r y o t y p i c d e v i a t i o n s w i t h i n c r e a s e i n growth advantage  (mixture  a d d i t i o n a l chromosomal c h a n g e s ) .  o f monosomy 22 w i t h and  accompanying  without  b.  Chromosomally Normal I n d i v i d u a l s — A b n o r m a l i t i e s With S p e c i f i c  Chronic myeloid retinoblastoma,  leukemia  A s s o c i a t i o n o f S p e c i f i c Chromosome  Malignancies  (CML), B u r k i t t lymphoma, meningioma,  and  a r e d i s e a s e s i n which s p e c i f i c chromosomal a b e r r a t i o n s  have been d e s c r i b e d i n the m a l i g n a n t  cells.  CML  i s considered to  a n e o p l a s t i c d i s e a s e o f the marrow i n which the major c l i n i c a l t i o n s r e l a t e t o abnormal, e x c e s s i v e , and  represent  manifesta-  apparently unrestrained  overpro-  duction of granulocytes.  A more d e t a i l e d d i s c u s s i o n o f t h i s d i s e a s e i s  g i v e n below  The  ( s e c t i o n 2) .  by Nowell and the l o n g arm  P h i l a d e l p h i a chromosome  H u n g e r f o r d i n 1960 o f chromosome #22  have a h e m o p o i e t i c  cell  (Ph"*") , f i r s t  (8), i s formed by a t r a n s l o c a t i o n i n v o l v i n g (9).  The  majority  l i n e which e x p r e s s e s  (>80%) o f CML  the Ph"'".  patients  In t h e v a s t m a j o r i t y  o f c a s e s , a l l o t h e r body t i s s u e s examined have t u r n e d out t o be somally normal. case  o f the m a l i g n a n t Ph^  i s found  chromo-  In no o t h e r known n e o p l a s t i c c o n d i t i o n i s t h e r e a  f o r the d i r e c t  involvement  state.  described  o f chromosomal change i n the  stronger  development  T h i s i s based on the c o n s i s t e n c y w i t h which the  i n the i n v o l v e d c e l l s v e r y e a r l y i n the d i s e a s e and,  generally,  i t s p e r s i s t e n c e throughout i t s course. In B u r k i t t lymphoma, n e o p l a s t i c c e l l s o f l y m p h o r e t i c u l a r d e r i v a t i o n occur  i n s o l i d tumors p r e d o m i n a n t l y  abdominal  i n the f a c i a l  ( n o n - A f r i c a n type) r e g i o n s .  ( A f r i c a n type) o r  In t h i s malignancy, t h e r e i s e v i -  dence f o r a s p e c i f i c chromosomal d e f e c t i n the m a l i g n a n t  cells.  In  most r e c e n t c y t o g e n e t i c study o f the n o n - A f r i c a n type o f B u r k i t t a p a r t i c u l a r t r a n s l o c a t i o n between chromosomes #8 and repeatedly  (10).  The  t r a n s l o c a t i o n was  tumor m a t e r i a l , o r i n c e l l unrelated p a t i e n t s .  The  found  #14  was  the  lymphoma,  found  i n d i r e c t preparations of  l i n e s d e r i v e d from tumor c e l l s ,  i n 12 out o f  same t r a n s l o c a t i o n has been f r e q u e n t l y found  in  18  -5-  s i m i l a r p r e p a r a t i o n s from the A f r i c a n type o f B u r k i t t lymphoma  (11).  Meningiomas a r e tumors composed o f s p e c i a l i z e d a r a c h n o i d a l cells.  lining  Chromosomal p r e p a r a t i o n s o f d i r e c t o r c u l t u r e d tumor m a t e r i a l from  over 200 meningiomas i n d i c a t e t h a t t h e s e tumors have a remarkable tendency to develop  h y p o d i p l o i d stem l i n e s .  d e l e t i o n o f chromosome #22 Retinoblastoma  PHA  i s a malignant  retinoblastoma  eye  One  step i s u s u a l l y a l o s s or  tumor o f c h i l d r e n which i s gene-  c y t o g e n e t i c study o f f i v e p a t i e n t s  showed normal k a r y o t y p e s  i n mitoses  from  s t i m u l a t e d p e r i p h e r a l b l o o d , but r e v e a l e d a s p e c i f i c chromosomal abnor-  m a l i t y i n c e l l s d e r i v e d from the tumor. that i n a l l f i v e cases, mitoses  prepared  d i r e c t l y from the tumor c e l l s . was  o f the D group chromosomes ( i . e . #13, Recently,  i n Ref.  13),  Apparently,  In f o u r o f the  #14,  or  and  These f o u r examples  of  one  o f tumor t i s s u e i n  reported  o f chromosome  #13.  t r a n s l o c a t e d onto chromosome  #20.  ( P h ^ - p o s i t i v e . CML,  retinoblastoma)  five  ( i n p r e p a r a t i o n and  a s m a l l d e l e t i o n i n the l o n g arm  t h e m i s s i n g m a t e r i a l was  the  chromosomally normal lymphocytes  In t h i s case, Balaban-Malenbaum  and n o n h e r e d i t a r y  reported  #15).  a more d e t a i l e d c y t o g e n e t i c examination  found  (12)  the d e l e t i o n o f the l o n g arm  a p a t i e n t with b i l a t e r a l retinoblastoma performed.  Hashem and K h a l i f a  abnormal chromosomes were d i s c o v e r e d i n some o f  p a t i e n t s , a c o n s i s t e n t abnormality  was  initial  (2).  t i c a l l y determined i n some c a s e s . with nonhereditary  The  B u r k i t t lymphoma, meningioma,  a r e t h e b e s t examples, t o . d a t e , o f human  n e o p l a s t i c , c o n d i t i o n s i n which t h e m a l i g n a n t  c e l l s have c h a r a c t e r i s t i c  chromosomal markers. Such s p e c i f i c i t y has  a l s o been demonstrated i n murine thymomas.  and h i s c o l l e a g u e s used chromosome b a n d i n g t e c h n i q u e s o f tumor c e l l s  from thymic lymphomas i n d u c e d  X - i r r a d i a t i o n , p o l y c y c l i c aromatic  t o study the  by t h r e e d i f f e r e n t  h y d r o c a r b o n s , and  Chan  karyotype  carcinogens:  endogenous leukemogenic  virus  (14).  Newborn mice o f two  89 tumors s t u d i e d , 76  (85%)  different  s t r a i n s were used.  Of a t o t a l o f  were c h a r a c t e r i z e d by t r i s o m y f o r chromosome  #15  (Thymus, s p l e e n , and bone marrow c e l l s were k a r y o t y p i c a l l y normal i n t h e s e animals.)  The  remaining  13 tumors were k a r y o t y p i c a l l y normal.  o b t a i n e d were independent used.  o f the c a r c i n o g e n i c agents  i n d u c e d i n mice c a r r y i n g t r a n s l o c a t i o n chromosomes i n v o l v i n g w i t h chromosomes #1,  #5,  o r #6,  d u p l i c a t i o n o f the gene(s) l o c a t e d on chromosome #15 tance  (15).  Thus, i n t h e s e R o b e r t s o n i a n  results  and t h e s t r a i n o f mouse  In a more r e c e n t r e p o r t , c y t o g e n e t i c s t u d i e s on T c e l l  f u s i o n o f chromosome #15  The  lymphomas centromeric  a l s o suggested  i s of c r i t i c a l  that  impor-  t r a n s l o c a t i o n s , even l a r g e chromo-  somes such as #1 were f o r c e d i n t o a s t a t e o f t r i s o m y a l o n g w i t h chromosome #15  i n a s i g n i f i c a n t number o f c a s e s . These five  examples of conditions  appear to be associated  with particular  in which unique chromosomal defects malignancies  in otherwise chromo-  somally normal individuals  have been invoked as support for  that a specific  in the genome,  change  c.  3  may  alteration  be a primary event in  observable  the  hypothesis  as a chromosomal  transformation.  Chromosomally Abnormal I n d i v i d u a l s Hereditary renal c e l l  carcinoma,  r e t i n o b l a s t o m a , and Wilms tumor are  examples o f d i s e a s e s i n which i n d i v i d u a l s w i t h a s p e c i f i c germ c e l l  mutation  v i s i b l e a t t h e chromosomal l e v e l i n a l l c e l l s ,  specific  t y p e o f malignancy.  are predisposed to a  In a d d i t i o n , i n d i v i d u a l s w i t h Down syndrome o r any  t h e chromosomal breakage syndromes a r e p r e d i s p o s e d t o d e v e l o p of  of  malignancies  a more g e n e r a l n a t u r e . Hereditary renal c e l l  unique  carcinoma  i s a malignancy  o f the k i d n e y .  In a  r e p o r t , Cohen and h i s a s s o c i a t e s d e s c r i b e d a f a m i l y i n which members  w i t h an i n h e r i t e d chromosomal t r a i t  were p r e d i s p o s e d t o r e n a l cancer  (16).  -7-  An a p p a r e n t l y b a l a n c e d t r a n s l o c a t i o n between chromosomes #3 and  #8 was  found  i n p e r i p h e r a l l e u k o c y t e s i n a l l e i g h t o f the p a t i e n t s w i t h r e n a l c a n c e r were k a r y o t y p e d .  No  The t r a n s l o c a t i o n was  f a m i l y member w i t h a normal k a r y o t y p e had t r a n s m i t t e d t o a p p r o x i m a t e l y one  offspring i n three generations. heterozygous pattern of  The  renal  h a l f o f the  who  cancer.  living  development o f r e n a l c a n c e r i n such  p e r s o n s w i t h the t r a n s l o c a t i o n f o l l o w e d an autosomal  dominant  inheritance.  In t h e p r e v i o u s s e c t i o n , the f i n d i n g o f a d e l e t i o n o f the l o n g arm chromosome #13  i n tumor c e l l s  r e t i n o b l a s t o m a was  noted.  from chromosomally normal i n d i v i d u a l s w i t h  Interestingly,  i n a minority of retinoblastoma  p a t i e n t s , c y t o g e n e t i c a n a l y s i s o f p e r i p h e r a l l e u k o c y t e s and  fibroblasts  r e v e a l e d i n t h e s e c e l l s a l s o the d e l e t i o n o f a s m a l l but s p e c i f i c o f the l o n g arm  o f chromosome #13  (2,17).  T h i s f i n d i n g has  t o suggest t h a t a p a r t i c u l a r r e g i o n on chromosome #13 mutation  of  may  has  segment  l e d some a u t h o r s  c a r r y t h e dominant  i n some i n s t a n c e s o f f a m i l i a l r e t i n o b l a s t o m a . (18), and t h a t  the  genes i n v o l v e d a t t h e somatic l e v e l a r e t h e same as t h o s e i n v o l v e d a t the germ c e l l l e v e l  (13).  Wilms tumor i s a k i d n e y c a n c e r t h a t a f f e c t s c h i l d r e n . associated with a n i r i d i a  (lack of i r i s e s ) ,  chromosomal a b n o r m a l i t y has been r e p o r t e d . the l i t e r a t u r e  and  I t i s sometimes  i n these cases, a c o n s i s t e n t  There  a r e a t l e a s t 18 c a s e s i n  (19) i n which a d e l e t i o n o f a s p e c i f i c band i n chromosome  has been r e p o r t e d t o e x i s t i n nontumor t i s s u e  (lymphocytes  and/or  b l a s t s ) , (20; Y u n i s and Ramsey, i n p r e s s and r e p o r t e d i n Ref.  fibro-  13).  These examples o f h e r e d i t a r y r e t i n o b l a s t o m a , h e r e d i t a r y r e n a l carcinoma,  and Wilms tumor, are t h e o n l y known i n s t a n c e s i n man  a s p e c i f i c chromosomal d e f e c t o c c u r r i n g p r e z y g o t i c a l l y appears these s u b j e c t s t o a s p e c i f i c tumor.  #11  cell  i n which to predispose  The chromosomal breakage syndromes comprise a number o f r a r e b u t distinct clinical  e n t i t i e s i n c l u d i n g F a n c o n i s anemia, Bloom syndrome, 1  a t a x i a t e l a n g i e c t a s i a , and xeroderma pigmentosum.  These g e n e t i c a l l y  determined syndromes have i n common an i n c r e a s e d tendency t o d e v e l o p m a l i g nancies i n general etc.) in  (acute leukemia, adenocarcinoma, squamous c e l l  carcinoma,  and a pronounced d i s p o s i t i o n t o spontaneous chromosomal breakage  vitro  Ref.  while r e t a i n i n g a d i p l o i d  s e t o f chromosomes  ( f o r a r e v i e w see  21). Down syndrome i s a c o n g e n i t a l d i s o r d e r , due t o t r i s o m y o f  #21.  The i n c i d e n c e o f b o t h . a c u t e m y e l o b l a s t i c and a c u t e  chromosome  lymphoblastic  leukemia i n i n d i v i d u a l s w i t h Down syndrome i s c l e a r l y h i g h e r than t h a t observed i n t h e g e n e r a l p o p u l a t i o n (22). These five further for  support  malignant  bility  examples of congenital  to the hypothesis  transformation.  that the association  malignant  transformation  predisposition  that genetic  Practical  In are  essential  to emphasize the  reveal  the primary  possiwith  event(s)  state.  Implications  c o n c l u s i o n , abnormal chromosome  not a r a r e o c c u r r e n c e .  c o n s t i t u t i o n s i n tumor t i s s u e  Indeed, a l t h o u g h t h e i r s i g n i f i c a n c e remains  unknown, t h e y a r e s u f f i c i e n t l y common t o have p r o v i d e d a u s e f u l to  lend  chromosomal abnormality  does not necessarily  that led to the development of a neoplastic  d.  change may be  They also serve of a particular  to cancer  the i d e n t i f i c a t i o n o f c e l l s belonging t o the malignant  approach  clone.  R e c e n t l y , some p r o g r e s s i n t h e c u l t u r i n g o f c e l l s from human tumor mens has been r e p o r t e d  (e.g. R e f s . 23 and 2 4 ) .  The use o f  speci-  chromosome  -9-  a n a l y s i s has been p a r t i c u l a r l y i m p o r t a n t c e l l s p r o l i f e r a t i n g in vitro  (25).  Nevertheless,  t h a t a l t h o u g h k a r y o t y p i c change may primary  event  mutation  may  s o m a l l y normal and  2.  The  remembered t o the  ( p r o b a b l y the m a j o r i t y ) the  initial  In such cases, changes v i s i b l e a t the chromosomal In t h i s s i t u a t i o n , a p p a r e n t l y chromo-  abnormal members o f t h e same m a l i g n a n t  the u t i l i t y  of karyotype  p o p u l a t i o n might  a n a l y s i s t o d i s t i n g u i s h normal  c e l l s would be c o r r e s p o n d i n g l y  and  limited.  M y e l o p r o l i f e r a t i v e Disorders  A number o f h e m a t o l o g i c  d i s o r d e r s appear t o r e p r e s e n t n e o p l a s t i c  c o n d i t i o n s r e s u l t i n g i n t h e o v e r p r o d u c t i o n o f one types  the  be so s m a l l t h a t i t i s not v i s i b l e by c o n v e n t i o n a l c y t o -  l e v e l would r e p r e s e n t l a t e r e v e n t s .  malignant  i t must be  accompany o r even c o r r e s p o n d  i n some c a s e s , i n o t h e r s  genetic techniques.  c o e x i s t , and  i n a s s e s s i n g the n a t u r e o f  t h a t . c o n s t i t u t e normal human b l o o d .  chronic myeloid thrombocytosis  leukemia (ET).  (CML), p o l y c y t h e m i a  In each case, t h e c e l l  o r more o f the  cell  Three such d i s o r d e r s a r e vera  (PV), and  essential  type p r e d o m i n a n t l y  i n the overproduction  i s different:  PV,  S i n c e a l l t h r e e d i s o r d e r s appear t o share a number o f  p l a t e l e t s i n ET.  biological  erythrocytes i n  f e a t u r e s , t h e y have been grouped t o g e t h e r under the term  m y e l o p r o l i f e r a t i v e d i s o r d e r s (MPD)  a.  g r a n u l o c y t e s i n CML,  involved  (26).  Time Course  All  t h r e e MPD  have i n common an e a r l y stage, which may  last  f o r years,  u s u a l l y c h a r a c t e r i z e d by a slow, p e r s i s t e n t i n c r e a s e i n the number and s e v e r i t y o f symptoms, sometimes accompanied by p e r i o d s o f  stability.  -10-  During t h i s phase, abnormal p r o l i f e r a t i o n  can u s u a l l y be h e l d i n check  by v a r i o u s forms o f t h e r a p y .  E v e n t u a l l y , i n PV and ET, t h e d i s e a s e may  p r o g r e s s t o an acute leukemic  s t a g e , and i n CML, t h i s i s t h e most common  result.  T h i s stage i s u n r e s p o n s i v e t o t h e r a p y and i s u s u a l l y  fatal  w i t h i n t h r e e t o s i x months.  b.  Single C e l l  Origin  A l t h o u g h t h e abnormal i n c r e a s e i n c i r c u l a t i n g b l o o d c e l l s i s u s u a l l y most pronounced  for a single c e l l  type  (i.e. red c e l l s  i n PV,  g r a n u l o c y t e s i n CML, p l a t e l e t s i n E T ) , t h e r e i s c o n s i d e r a b l e e v i d e n c e t o i n d i c a t e that the primary d e f e c t l i e s i n a c e l l capable o f g i v i n g r i s e t o a l l three c e l l (Ph'S  lineages.  F o r example, i n CML t h e P h i l a d e l p h i a chromosome  has been found i n e r y t h r o i d , g r a n u l o c y t i c ,  i n i n d i v i d u a l s who a r e o t h e r w i s e chromosomally the predominant of  malignant c e l l  and m e g a k a r y o c y t i c  normal  type i s g r a n u l o c y t i c .  P h i ^ - p o s i t i v e CML p a t i e n t s who were h e t e r o z y g o u s  A and B a l l e l e s  (27), even  though  Subsequent  studies  f o r t h e X - l i n k e d G6PD  s t r o n g l y support the hypothesis that these d i s e a s e s , l i k e  many o t h e r neoplasms, a r i s e  from a s i n g l e c e l l  from which a c l o n e o f : c e l l s  w i t h s u p e r i o r growth o r s u r v i v a l p o t e n t i a l p r o l i f e r a t e s t i o n , t h e y have c o n f i r m e d t h e n o t i o n t h a t t h i s c e l l pluripotent  stem c e l l  (28,29).  p a t i e n t s heterozygous  In addi-  i s a member o f t h e  compartment.  PV has a l s o been shown t o be o f u n i c e l l u l a r o r i g i n u s i n g  dominant c e l l  cells  f o r two G6PD a l l e l e s  (30) and even though  female the p r e -  type i s e r y t h r o i d , g r a n u l o c y t e s and p l a t e l e t s a l s o show a  s i n g l e enzyme marker.  T h i s s u g g e s t s t h a t i n PV, l i k e CML, t h e p r i m a r y  defect i s i n a single c e l l  capable o f g i v i n g r i s e to a l l three c e l l  types.  -11-  c.  Chromosome S t u d i e s  There i s a c o n s i d e r a b l e amount o f i n f o r m a t i o n a v a i l a b l e on involvement  o f chromosomal changes i n t h e MPD.  t h i s group r e p r e s e n t s 70% i n v e s t i g a t e d to date  the  Mitelman e s t i m a t e s  that  o f the 4,000 human tumors c y t o g e n e t i c a l l y  (31) .  The  r o l e o f t h e Ph"*" i n CML  has  a l r e a d y been  mentioned. Up  t o 26% o f u n t r e a t e d PV p a t i e n t s have some chromosomal abnor-  m a l i t y i n t h e i r bone marrow c e l l s a t the time o f d i a g n o s i s most common chromosomal a b n o r m a l i t i e s i n u n t r e a t e d PV chromosomes i n group C  ( i . e . #6 t o #12)  most f r e q u e n t a b n o r m a l i t y abnormality (36).  (37) .  In t r e a t e d PV,  T h i s chromosomal a b n o r m a l i t y  the  the most common o f chromosome  #20  a s s o c i a t e d w i t h ET.  Among n e a r l y 30 c a s e s  R a j e n d r a e t a l . have s t u d i e d one  The  MPD  (41).  The  as M a l i g n a n t  Although t h e y can be  be  s p e c i f i c chromosomal a b n o r m a l i t y  t o be c y t o g e n e t i c a l l y normal  marrow c e l l s  may  r e l a t e d t o the  (2).  There i s no  the MPD  y e t known t o  be  r e p o r t e d , t h e m a j o r i t y were  (2,38,39,40).  In a  unique•report,  ET p a t i e n t w i t h Ph"*" i n 90%  o f the bone  s i g n i f i c a n c e o f t h i s remains u n c l e a r .  Conditions  have i n common a p r o l o n g e d  phase d u r i n g which  f a i r l y w e l l c o n t r o l l e d , t h e y have f e a t u r e s c o n s i s t e n t w i t h  t h e i r being considered malignancies. of  appear t o i n v o l v e  w i t h an e x t r a #8 o r #9 b e i n g  appears t o be a d e l e t i o n o f the l o n g arm  g e n e s i s o f PV  d.  The  Rare cases o f t h i s d e l e t i o n have been e n c o u n t e r e d i n u n t r e a t e d  patients  found  (33,34,35).  (32).  a s i n g l e p l u r i p o t e n t stem c e l l  They a r i s e from t h e  (29,30) g i v i n g r i s e t o a  transformation continuously  -12-  expanding  clone of c e l l s r e l a t i v e l y  i n s e n s i t i v e t o normal r e g u l a t i o n  c a p a b l e o f s u p p r e s s i n g the growth o f r e m a i n i n g normal c e l l s . f e a t u r e o f a c h r o n i c s t a g e , which may the acute leukemic  stage, make the MPD  i d e a l model systems f o r the  b e h a v i o u r , t h a t are p a r t o f an o n g o i n g m a l i g n a n t  The  Hemopoietic  Figure 1).  study  cellular  transformation process.  System  The m a j o r i t y o f c e l l s t h a t c i r c u l a t e ferentiated,  The  l a s t up t o s i x y e a r s b e f o r e  o f g e n e t i c f e a t u r e s , i . e . chromosomal changes and a l t e r e d  3.  and  incapable of p r o l i f e r a t i o n ,  i n the b l o o d a r e h i g h l y  and r e l a t i v e l y  Current concepts o f hemopoiesis  h i e r a r c h y o f committed p r o g e n i t o r c e l l  short-lived  dif(see  f a v o u r the e x i s t e n c e o f a  t y p e s f o r each pathway w i t h p r o -  g r e s s i v e l y d e c r e a s i n g c a p a c i t y f o r p r o l i f e r a t i o n and  limited,  and  i f any,  self-renewal capacity.  E r y t h r o i d , megakaryocytic,  granulopoietic  p r o g e n i t o r s a r e thought  t o have, i n t u r n , a common o r i g i n from a  pluri-  p o t e n t stem c e l l which i s c a p a b l e o f s e l f - r e n e w a l but r e s t r i c t e d t o myelopoiesis.  There  i s a l s o now  some e v i d e n c e  f o r the e x i s t e n c e o f a more  " p r i m i t i v e " stem c e l l t h a t can g i v e r i s e t o lymphoid progeny.  The  as w e l l as m y e l o i d  experimental data s u p p o r t i n g these concepts are d i s c u s s e d  below.  a.  Colony Assays F o r P r i m i t i v e C e l l  The  Types  f a c t that p r i m i t i v e hemopoietic  c e l l s a r e r a r e and not morpho-  l o g i c a l l y d i s t i n c t has l e d i n v e s t i g a t o r s t o use c l o n o g e n i c a s s a y s t o i d e n t i f y them.  In 1961,  Till  and M c C u l l o c h  c e l l s , when i n j e c t e d i n t o h e a v i l y i r r a d i a t e d  showed t h a t mouse marrow s y n g e n i c mice, were c a p a b l e  Figure 1.  The hemopoietic system. A schematic illustration of stem c e l l differentiation showing lymphoid and myeloid development from a common pluripotent stem c e l l . ,  -14-  o f forming d i s c r e t e n o d u l e s o f h e m a t o p o i e t i c c e l l s i n the animals e i g h t to t e n days a f t e r i n j e c t i o n  (42).  spleens  These n o d u l e s were  found t o c o n t a i n e r y t h r o i d , g r a n u l o c y t i c , m e g a k a r y o c y t i c , and tiated cells,  e i t h e r as pure p o p u l a t i o n s  D e f i n i t i v e proof  o f these  undifferen-  or i n v a r y i n g m i x t u r e s  (43).  o f the c l o n a l n a t u r e o f t h e s e nodules.was p r o v i d e d  s t u d i e s i n which marrow c e l l s c o n t a i n i n g  specific  by  s t a b l e chromosomal  markers were i n j e c t e d i n t o i r r a d i a t e d , but o t h e r w i s e chromosomally normal recipients. 99%  Some c o l o n i e s showed u n i q u e l y  o f the metaphases  c o l o n i e s had  (44).  i r r a d i a t e d mouse and Using  That the c e l l s  s e l f - r e n e w a l p o t e n t i a l was  pension of c e l l s derived  abnormal k a r y o t y p e s i n 95 that  demonstrated by  from a s p l e e n c o l o n y  observing  t h i s method, c e l l s  new  spleen  i n apparently  gave r i s e  pure s p l e e n  colonies  marrow c e l l  three' m y e l o i d c e l l  that t h i s colony  and  ate and d i f f e r e n t i a t e e x t e n s i v e l y and S t u d i e s by Wu  and  a p l u r i p o t e n t stem c e l l lymphoid c e l l s  (48).  lethally  ( i . e . only  capable of giving  types a f t e r r e t r a n s p l a n t a t i o n  These s t u d i e s showed t h a t one types,  sus-  c o l o n i e s t e n t o 14 days l a t e r (45).  g r a n u l o c y t i c o r e r y t h r o i d c e l l s seen) were shown t o be r i s e to colonies of a l l c e l l  to these  injecting a  i n t o another  to  (46,47).  c o u l d form a c o l o n y  undergo  associates provided  containing a l l  forming-cell could  prolifer-  self-renewal. support  f o r the existence  capable of g i v i n g r i s e to both myeloid  of  and  These i n v e s t i g a t o r s showed t h a t when i r r a d i a t e d  mice were t r a n s p l a n t e d w i t h marrow c o n t a i n i n g unique  radiation-induced  chromosomal, markers, the marker chromosomes were s u b s e q u e n t l y found i n the lymphoid as w e l l as m y e l o i d t i s s u e s . colleagues  reported  his  a case o f s i d e r o b l a s t i c anemia i n a human female  h e t e r o z y g o u s f o r G6PD A and in erythrocytes,  More r e c e n t l y , P r c h a l and  B a l l e l e s who  granulocytes,  showed the  same, s i n g l e isoenzyme  and macrophages, as w e l l as B and  T  -15-  lymphocytes,  s u g g e s t i n g an o r i g i n o f b o t h m y e l o i d and  from a s i n g l e c e l l  (49).  o f b o t h isoenzymes  i n h e r f i b r o b l a s t s and c e l l u l a r s a l i v a r y  b.  In  Vitro  G6PD h e t e r o z y g o s i t y was  lymphoid  cells  shown by the p r e s e n c e lysates.  Assays F o r Hemopoietic P r o g e n i t o r s  In vitro  c o l o n y assays have been d e v e l o p e d t h a t p e r m i t the growth  and m a t u r a t i o n o f c e l l s t h a t g i v e r i s e t o e r y t h r o c y t e s , g r a n u l o c y t e s , and macrophages  ( f o r a review see Ref. 5 0 ) .  The  f a c t t h a t these p r i m i t i v e p r e -  c u r s o r s a r e p r e s e n t a t low f r e q u e n c y and do not p o s s e s s d i s t i n c t morphological  features precludes t h e i r d i r e c t recognition.  c o l o n i e s c o n t a i n i n g o n l y one  The development o f  c e l l l i n e a g e (e.g. e r y t h r o i d c e l l s )  i s dependent  on the a d d i t i o n t o t h e c u l t u r e medium o f s p e c i f i c s t i m u l a t o r y f a c t o r s .  Such  "pure" c o l o n i e s are thought t o r e p r e s e n t the c l o n a l descendants  of  p r o g e n i t o r s s i n c e they a r i s e i n c u l t u r e s i n which more than one  c o l o n y type  i s obtained. The  (See T a b l e I f o r a l i s t o f the c o l o n y assays  a s s a y s i n v o l v e suspending h e m o p o i e t i c c e l l s  "committed"  available.)  i n one o f v a r i o u s  t y p e s o f s e m i s o l i d media c o n t a i n i n g t h e a p p r o p r i a t e n u t r i e n t s , serum, and stimulatory  factors.  A f t e r one t o two weeks o f i n c u b a t i o n , e r y t h r o i d  colonies  can be i d e n t i f i e d by t h e i r r e d c o l o u r , i n d i c a t i v e o f ongoing hemoglobin t h e s i s i n e r y t h r o i d c e l l s t h a t have reached t h e i r tion.  f i n a l stage o f  differentia-  E r y t h r o i d c o l o n i e s composed o f 16 o r more c l u s t e r s o f c e l l s ,  c l u s t e r c o n t a i n i n g about 50 c e l l s , a r e thought t o r e p r e s e n t the  syn-  each  descendants  o f the most p r i m i t i v e c e l l s which are committed t o the e r y t h r o i d l i n e a g e (66). The r e l a t i o n between the p r o l i f e r a t i v e  c a p a c i t y o f the o r i g i n a l p r o g e n i t o r  c e l l and t h e t i m e taken t o generate hemoglobin  producing erythroblast  s u g g e s t s t h a t t h e s i z e o f c o l o n y formed in vitro  i s determined by the  o f d i f f e r e n t i a t i o n o f the c o l o n y forming c e l l in vivo  (72).  progeny, state  -16-  T a b l e I.  S p e c i f i c Stimulating Factors f o r Myeloid  Species  Colony Type  GranulocyteMacrophage  Megakaryocyte  Eosinophil  Small E r y t h r o i d  Mouse  (51,52)  Sources o f Presumed Stimulating Factors  Human u r i n e (53) Mouse f i b r o b l a s t c e l l medium (54)  conditioned  Human l e u k o c y t e medium (56)  Mouse (57)  Mitogen s t i m u l a t e d mouse s p l e e n c e l l c o n d i t i o n e d medium (58)  Man (59)  E r y t h r o p o i e t i n from anemic plasma  Mouse (60)  Mitogen s t i m u l a t e d mouse s p l e e n c e l l c o n d i t i o n e d medium (58)  Man (56)  Human l e u k o c y t e medium (56)  Mouse (61)  E r y t h r o p o i e t i n from anemic plasma E r y t h r o p o i e t i n from anemic plasma  Mouse  Man  Mixed  Lineages.  Man (55)  Man (62) Large E r y t h r o i d  Cell  (63,64)  (66,67)  Mouse  (69,70)  Man (71)  conditioned  sheep  conditioned  sheep sheep  E r y t h r o p o i e t i n and mitogen stimulated spleen c e l l conditioned medium (65) E r y t h r o p o i e t i n and human l e u k o c y t e c o n d i t i o n e d medium (68) E r y t h r o p o i e t i n and mitogen s t i m u l a t e d mouse s p l e e n c e l l c o n d i t i o n e d medium E r y t h r o p o i e t i n and mitogen stimulated leukocyte conditioned medium  T h i s l i s t i s n o t meant t o be a l l inclusive,- s o u r c e s named a r e commonly used examples. Numbers i n b r a c k e t s i n d i c a t e r e f e r e n c e s .  -17-  Under i d e a l c u l t u r e c o n d i t i o n s , g r a n u l o p o i e t i c c o l o n i e s can be  f a i r l y r e l i a b l y i d e n t i f i e d w i t h o u t f i x a t i o n and  t h e i r c h a r a c t e r i s t i c morphology and Several derived  l a c k of red colour  colony  forming c e l l s ,  and  c e l l s and  These i n c l u d e the m i c r o m a n i p u l a t i o n the  subsequent o b s e r v a t i o n  raphy o f the d e v e l o p i n g cells  (50).  of colonies  c o l o n i e s . (74) ; and,  i n the c u l t u r e d i s h by p l a s t i c  rings  same pathway o f  of s i n g l e colony  Chronic  techniques  the  76),  but  on c u r r e n t c o n c e p t s o f hemopoiesis, one  t h e s e g i v e no  A n a l y s i s o f c e l l s a t t h i s end  the f a c t t h a t t h e s e d i s e a s e s  p l a s t i c transformation  event p r o b a b l y  have a l r e a d y been mentioned. bone marrow c e l l s  i n these d i s e a s e s  rela-  stage o f MPD  progression.  i n myeloid  cells  have i n common a neo-  o c c u r r i n g i n a m y e l o i d stem  Karyotypic  of  from  g i v e an i n c o m p l e t e p i c t u r e i n the case o f the  s p e c i f i c i t y o f chromosomal a b n o r m a l i t i e s  amongst the MPD, and  Based  would expect the m a j o r i t y  where k a r y o t y p e e v o l u t i o n i s known t o accompany d i s e a s e The  information  from a bone marrow a s p i r a t e t o be d e r i v e d  t i v e l y mature e r y t h r o c y t e s . d i f f e r e n t i a t i o n may  Colonies  are making i t e a s i e r t o o b t a i n banded k a r y o t y p e s  s t a t e o f d i f f e r e n t i a t i o n o f the c e l l k a r y o t y p e d .  karyotypes obtained  the  (CML)  from human bone marrow (e.g. Ref. regarding  photog-  (75).  a.  New  forming  p h y s i c a l i s o l a t i o n of  A p p l i c a t i o n o f Chromosomal A n a l y s i s t o S i n g l e Hemopoietic  Leukemia  these  differ-  (73); t i m e - l a p s e  4.  Myeloid  clones  t h a t the p r o g e n i e s o f  c e l l s are i r r e v e r s i b l y committed t o remain i n the entiation.  s t a i n i n g by v i r t u e o f  l i n e s o f e v i d e n c e i n d i c a t e t h a t the c o l o n i e s are  from single  also  cell,  a n a l y s i s of f r e s h l y i s o l a t e d  i s a recognized  tool in their  -18-  differential suggests  d i a g n o s i s and treatment  s i n g l e c o l o n i e s o f hemopoietic r e s i d u a l normal stem c e l l s  significant  cells  However, t h i s t y p e o f a n a l y s i s w i l l  t o d e t e c t even a l a r g e , but q u i e s c e n t o r suppressed,  chromosomally normal stem c e l l s .  of  information  t h a t t h e p e r c e n t a g e o f chromosomally abnormal marrow  increases as the disease progresses. fail  (77) , and c u r r e n t  population of  A n a l y s i s o f chromosomes o b t a i n e d  from  c e l l s p r o v i d e s an a p p r o a c h t o t h e q u e s t i o n i n t h e MPD.  importance i n d e t e r m i n i n g  Such a f i n d i n g c o u l d be o f  the treatment  o f choice i n a given  patient. T h e r e i s some evidence  t o support  the hypothesis  normal, nonmalignant, stem c e l l s may p e r s i s t Chervenick  t h a t chromosomally  i n p a t i e n t s with  MPD.  and a s s o c i a t e s r e p o r t e d f i n d i n g chromosomally normal hemo-  p o i e t i c c o l o n i e s i n P h ^ - p o s i t i v e CML (78) .  More r e c e n t l y , S i n g e r and h i s  c o l l e a g u e s s t u d i e d a P h ^ - p o s i t i v e CML p a t i e n t , who was a l s o heterozygous for  t h e A and B forms o f G6PD, b e f o r e and a f t e r i n t e n s i v e chemotherapy  P r i o r t o treatment, platelets,  o n l y one G6PD isoenzyme was p r e s e n t  and g r a n u l o c y t e s ,  phases were P h ^ - h e g a t i v e .  i n r e d blood  (30) .  cells,  and o n l y a s m a l l p e r c e n t a g e o f marrow meta-  A f t e r t h r e e months o f i n t e n s i v e chemotherapy,  isoenzymes were d e t e c t e d i n t h e b l o o d c e l l s , marrow metaphases were P h ^ - n e g a t i v e .  both  and t h e m a j o r i t y o f t h e  These p r e l i m i n a r y d a t a i n d i c a t e d  t h a t w i t h c o n v e r s i o n t o Ph"*" n e g a t i v i t y a f t e r i n t e n s i v e chemotherapy, n o n c l o n a l and, presumably, n o n n e o p l a s t i c  b.  Polycythemia  hematopoiesis  was r e s t o r e d .  V e r a (PV)  Cytogenetic  a n a l y s i s o f s i n g l e hemopoietic  c o l o n i e s from  individuals  w i t h PV p r o v i d e s a unique o p p o r t u n i t y t o compare t h e chromosomal f i n d i n g s i n  -19-  two  apparent  populations  nant t r a n s f o r m a t i o n Erythropoietin colonies  o f stem c e l l s i n a d i s e a s e  i s thought t o o c c u r i n a s i n g l e m y e l o i d  (epo)  i s a hormone r e q u i r e d  from normal i n d i v i d u a l s .  p a t i e n t s can colonies  be  that  do not  f o r maturation i n  (66,79,80,81).  w i t h o u t epo,  dependent e r y t h r o i d c o l o n i e s may  the r e l e v a n c e be determined.  chromosomes were found o n l y i n the  obtained  o f the p e r c e n t a g e o f  be  t h a t the o b s e r v a t i o n  i n d i c a t i v e o f the In PV,  as  existence  i n CML,  important t h e r a p e u t i c  the  epo-independent e r y t h r o i d c o l o n i e s ,  o f normal m y e l o i d stem  existence  then by  derived  simply  from a PV p a t i e n t , one  normal m y e l o i d stem  c.  counting  Present  I f chromosomal s t u d i e s  importance o f b e i n g  can  support  the  from normal stem colonies  o b t a i n a q u a n t i t a t i v e measure o f  the  Objective  s e c t i o n s , arguments were p r e s e n t e d t h a t able  t o a n a l y z e the  l e v e l of p r i m i t i v e hemopoietic c e l l cell  has  cells.  In the p r e c e d i n g the  stem c e l l s  the numbers o f epo-dependent can  should  cells.  o f normal m y e l o i d  h y p o t h e s i s t h a t the epo-dependent c o l o n i e s are d e r i v e d cells,  malignant  o f epo-dependent e r y t h r o i d c o l o n i e s  implications.  epo-  I f , f o r example, abnormal  then t h i s would suggest t h a t epo-independence i s a marker f o r the clone, and  PV  of erythroid  comparing chromosomal f i n d i n g s i n e r y t h r o i d c o l o n i e s  i n c u l t u r e s w i t h and  cell.  Recent r e p o r t s have shown t h a t  r e q u i r e normal l e v e l s o f epo  cultures  stem  malig-  f o r the m a t u r a t i o n o f e r y t h r o i d  c h a r a c t e r i z e d by h a v i n g a h i g h p r o p o r t i o n  methylcellulose By  i n which the  o n l y be  indicate  chromosomal p i c t u r e a t  differentiation.  Since  the  t h i s type o f  i d e n t i f i e d by a s s a y s t h a t p e r m i t c l o n a l e x p a n s i o n  -20-  and m a t u r a t i o n , the main o b j e c t i v e method f o r o b t a i n i n g hemopoietic  colonies.  i n t h i s work was t o d e v e l o p a r e l i a b l e  h i g h q u a l i t y G- and Q-banded k a r y o t y p e s from s i n g l e  -21-  MATERIALS AND METHODS  1.  P a t i e n t Specimens P e r i p h e r a l b l o o d and bone marrow samples were o b t a i n e d through t h e  courtesy o f p h y s i c i a n s a t the f o l l o w i n g i n s t i t u t i o n s :  Vancouver  General  H o s p i t a l , P r i n c e George G e n e r a l H o s p i t a l , Royal Columbian H o s p i t a l (New Westminster), tories  Royal J u b i l e e H o s p i t a l ( V i c t o r i a ) ,  (Victoria),  and L i o n s Gate H o s p i t a l (North V a n c o u v e r ) .  patient information, including c l i n i c a l of  Island Medical  f o r which i t was used,  i n T a b l e I I . A l l samples were l e f t o v e r specimens t a k e n w i t h f o r t h e purpose o f c l i n i c a l  Relevant  d i a g n o s i s and t r e a t m e n t ,  specimen o b t a i n e d , and t h e experiment  consent  Labora-  t h e type a r e shown  informed  diagnosis, staging, or follow-up.  F i v e a d d i t i o n a l normal a d u l t p e r i p h e r a l b l o o d specimens were c o l l e c t e d from l o c a l v o l u n t e e r s .  2.  Specimen P r e p a r a t i o n Bone marrow a s p i r a t e from t h e s u p e r i o r p o s t e r i o r i l i a c c r e s t , and  p e r i p h e r a l b l o o d t a k e n by v e n i p u n c t u r e , were c o l l e c t e d i n p r e s e r v a t i v e free heparin  (Connaught L a b o r a t o r i e s ) , 400 units/marrow specimen and  50 u n i t s / m l b l o o d t o p r e v e n t c l o t  formation.  marrow a s p i r a t e v a r i e d from 1 t o 5.5 ml.  The volume o f t h e bone .  P e r i p h e r a l b l o o d specimens were  c o l l e c t e d i n volumes o f 10 ml. Marrow c e l l s were p r e p a r e d and Eaves  ( 6 6 ) . Marrow b u f f y c e l l s were o b t a i n e d r e l a t i v e l y f r e e o f r e d  c e l l s u s i n g a two-step p r o c e d u r e to  f o r p l a t i n g u s i n g t h e method o f Gregory  t h r e e minutes,  o f c e n t r i f u g a t i o n a t 1,000 rpm f o r two  f o l l o w e d by s e d i m e n t a t i o n o f t h e b u f f y c o a t a t u n i t  g r a v i t y f o r t e n t o 20 minutes. and t h e plasma d i s c a r d e d .  The c e l l r i c h plasma was then r e c e n t r i f u g e d  Finally,  c e l l s were washed once i n a-medium  Table I I .  Patient Data; SOUJCCS  Age  Diagnosis  of  *  Subject  Sex  .  Treatment  ID  M  23  Normal  PB  KH  M  31  Normal  PB  Nil  HS  M  31  Normal  M  Nil  AJ  M  31  CML  M  C&R  IH  F  52  Acute myelofibrosis  MD  M  27  Lymphoma  TS  F  ' 43  CML  PC  M  61  Stress erythrocytosis  NF  F  65  PV  M  HS  M  31  Normal  M  Nil  QL  M  58  Plasmacytoma  M.  Nil  WK.  M  59  2° Erythrocytosis  M  Nil  DL  F  60  Myeloma  M  Nil  JB '  F  9  GK  M  NN  Nil  M  C&R  M  Nil •  .R SCE studies  Bloom syndrome  PB&M  Nii  35  Normal  PB  Nil  F  45  Normal  PB  Nil  KN  M .  41  PV  PB  LD  F  8  JM  F  62  PV  HK  M  56  ET  FN  M  58  Trisomy 8  Myeloma  Time course studies  Nil  PB  PB  Experiment  Nil  PB  Nil  PB&M  Nil  M  Nil  M  Nil  Karyotypic  studies  (continued...)  Table I I .  (continued) Age -  Source of „ . Specimen  „ , * Treatment  Subiect J  Sex  RR  M  40  CML .  PB  C  GH  F  52  ET  M  Nil  IM  F  18  CML  PB  C  CF  F  64  2° Erythrocytosis  M  Nil  WH  M  30  PV  PB  Nil  CF  M  63  CML  M  Nil  RH  M  77  PV  RAl  F  52  Lymphoma  WK  M  59  EM  F  56  SH  F  37  SL  F  KS  M  47  NF  F  65  WC  M  74  2° Polycythemia  RA2  M  43  .. Erythrocytosis  PB&M  Nil  AE  M  38  Normal  PB  Nil  PD  M  64  . PV  M  Nil  HW  M  26  CML  PB  Nil  HC  F  71  ET  M  Nil  0  Diagnosis -  Patient Data.  3  PB&M M  Nil Nil  Normal  PB&M  Nil  PV  PB&M  Nil  CML  PB  C  Lung cancer  PB&M  R  PB&M  Nil  PV  M  Karyotypic studies  Nil  PB&M  2° Erythrocytosis  „ . Experiment  C  (continued...)  Table II.  (continued)  Patient Data. *  Subject  Sex  Age  Diagnosis  Source of Specimen  Treatment  Experiment  NF  F  65  PV  PB&M  Nil  Karyotypic studies  WS  M  24  Hodgkins lymphoma  M  R  AM  M  91  Myeloma  M  Nil  OD  F  52  PV  PB  C  CD  M  61  Myeloma  M  Nil  Abbreviations used:  II  it  PB, peripheral blood; M, bone marrow; C, chemotherapy; R, radiotherapy; CML, chronic myeloid leukemia; PV, polycythemia vera; ET, essential  Within 6 months.  II  thrombocytosis.  (Connaught L a b o r a t o r i e s concentration  o f 2%  (For the e x a c t  1403-01-07) c o n t a i n i n g  (2% FCS)  and  resuspended i n the  d e t a i l s o f the p r e p a r a t i o n  P e r i p h e r a l blood  fetal calf  serum a t a  final  same f o r f i n a l p l a t i n g .  p r o c e d u r e , see Appendix  1A.)  specimens were p r o c e s s e d u s i n g F i c o l l - h y p a q u e  (LSM  B i o n e t i c s B410-01) d e n s i t y c e n t r i f u g a t i o n t o i s o l a t e the mononuclear f r a c t i o n according FCS  and  t o the m a n u f a c t u r e r ' s d i r e c t i o n s .  then f i n a l l y resuspended i n the C e l l s were p l a t e d i n 35 x 1 0  (Lux  #5221-R) i n 1.1  0.8%  methylcellulose,  specimens), 0.1%  mm  C e l l s were washed t w i c e i n  same f o r p l a t i n g (see Appendix  standard  ml o f c u l t u r e medium. 30%  f e t a l c a l f serum  deionized  nontissue  The  IB).  culture petri  dishes  f i n a l medium c o n s i s t e d  (the. same l o t was  2%  of  used f o r a l l  b o v i n e serum albumin, 9% human l e u k o c y t e  condi-  -4 t i o n e d medium (epo,  (66), 10  M 2-mercaptoethanol, 2.5  Step I I I , Connaught L a b o r a t o r i e s ) , and  i n 2% FCS  (see Appendix 1C).  final cell  concentration  preparations  10%  units/ml o f the  Bone marrow p r e p a r a t i o n s  of  erythropoietin  final cell  suspension  were p l a t e d a t a  o f • 2 x 10.^ c e l l s p e r .dish,-while, p e r i p h e r a l  were p l a t e d a t 4 x 1 0 ^  C u l t u r e s were i n c u b a t e d maintained at high humidity.  c e l l s per  blood  dish.  a t 37°C i n a 5% C 0  2  i n a i r environment  D i s h e s were examined v i s u a l l y ,  u s i n g an  and inverted  m i c r o s c o p e , a f t e r e i g h t o r n i n e days, f o r the p r e s e n c e o f e r y t h r o p o i e t i c colonies.  These can be r e a d i l y and  types of c o l o n i e s present globin synthesis. reddish tinge and m i t o t i c 3.  in living  When t h i s o c c u r s ,  (72).  The  day  s p e c i f i c a l l y d i s t i n g u i s h e d from  other  c u l t u r e s o n l y a f t e r the o n s e t o f hemoerythroid colonies acquire  of harvest  was  a  distinct  chosen t o maximize c o l o n y  size  index.  Chromosome H a r v e s t The  method o f Moorhead and  hemopoietic c o l o n i e s .  One  h i s colleagues  hour p r i o r t o the  end  (82)  was  o f the  a p p l i e d to  incubation  the  period,  -26-  c e l l d i v i s i o n was a r r e s t e d a t metaphase by the a d d i t i o n o f 0.12 ml c o l c e m i d (Gibco #120-5210) t o each 1.1 ml c u l t u r e  ( f i n a l c o n c e n t r a t i o n , 0.1  ug/ml).  The c o l c e m i d was d i l u t e d t o 1 yg/ml i n Hanks BSS and then a p p l i e d u s i n g a 26-gauge s y r i n g e n e e d l e t o ensure even a p p l i c a t i o n o v e r the s u r f a c e o f t h e methylcellulose. Pasteur p i p e t t e microscope  S i n g l e c o l o n i e s were p l u c k e d i n t o a f i n e l y drawn o u t ( i n t e r n a l d i a m e t e r o f a p p r o x i m a t e l y 0.4 mm)  and f i n a l m a g n i f i c a t i o n 75x  (see F i g u r e 2 ) .  u s i n g an i n v e r t e d  The c o l o n y ,  suspended  i n a p p r o x i m a t e l y 0.01 ml o f growth media, was t h e n t r a n s f e r r e d t o a c o n i c a l p o l y p r o p y l e n e c e n t r i f u g e tube  ( c a p a c i t y 0.5 ml, E v e r g r e e n 3403) c o n t a i n i n g  0.4 ml o f 0.075 M KC1 a t room t e m p e r a t u r e .  Clumps i n t h e c o l o n y were broken  up by g e n t l y b u b b l i n g a i r through t h e h y p o t o n i c s o l u t i o n w i t h a f i n e l y drawn o u t p i p e t t e .  The c e l l  s u s p e n s i o n was then i n c u b a t e d f o r 20 minutes  a t room temperature  before c e n t r i f u g a t i o n .  To f a c i l i t a t e c e n t r i f u g a t i o n , i n t o l a r g e r tubes  t h e s m a l l c o n i c a l tubes were p l a c e d  ( 1 7 x 1 0 0 mm p l a s t i c d i s p o s a b l e t u b e s , F a l c o n 2057), and  then spun a t 800 rpm  (~200 g) f o r e i g h t minutes.  A t t h e end o f c e n t r i f u g a -  t i o n , a l l but 0.05 ml o f t h e s u p e r n a t a n t was removed by a s p i r a t i o n and t h e c e l l s were resuspended 0.4 ml o f f i x a t i v e  i n the r e m a i n i n g KC1 by g e n t l y b u b b l i n g w i t h a i r .  (3:1 a b s o l u t e m e t h y l a l c o h o l : g l a c i a l a c e t i c a c i d ) was  then added t o t h e s m a l l c e n t r i f u g e tube and the e n t i r e c o n t e n t s mixed by gently bubbling a i r .  After at least  20 minutes,  the c e l l s were spun down  as b e f o r e and a l l but 0.05 ml o f t h e s u p e r n a t a n t was d i s c a r d e d . washed i n 0.4 ml o f f r e s h f i x a t i v e , c a r d e d as b e f o r e .  c e n t r i f u g e d , and t h e s u p e r n a t a n t  dis-  T h i s washing p r o c e d u r e was r e p e a t e d b e f o r e t h e c e l l s  were f i n a l l y suspended The  C e l l s were  final cell  i n 0.05 ml o f f i x a t i v e . s u s p e n s i o n was c a r e f u l l y drawn up i n t o a f i n e l y drawn  out p i p e t t e and dropped square No. 1 c o v e r g l a s s  from a h e i g h t o f about (Corning).  1 cm onto a c l e a n , d r y , 22 mm  The c e l l s were s p r e a d by g e n t l y b l o w i n g ,  -27-  (C)  Figure  2.  Colony plucking. Removal o f a s i n g l e h e m o p o i e t i c c o l o n y ( e s t i m a t e d s i z e o f 1,500 c e l l s ) u s i n g a f i n e l y drawn o u t Pasteur p i p e t t e .  and t h e c o v e r g l a s s was d r i e d by h o l d i n g i t 5 cm from a 150 watt grow lamp ( S y l v a n i a spot-grow)  4.  Chromosome Banding The G-banding  and a s s o c i a t e s old, for  f o r 30 seconds.  t e c h n i q u e used was a m o d i f i c a t i o n o f t h a t o f Chandhuri  (83). A i r d r i e d , u n s t a i n e d c o v e r g l a s s e s , a t l e a s t 24 hours  were immersed i n pH 6.8 phosphate b u f f e r  (BDR Chemicals 702002) a t 65°C  one hour, and then s t a i n e d i m m e d i a t e l y i n 30% W r i g h t s s t a i n  (Matheson,  Coleman, and B e l l Inc.) i n pH 6.8 phosphate b u f f e r f o r 45 seconds. g l a s s e s were mounted on s t a n d a r d m i c r o s c o p e s l i d e s i n 25% E u k i t t media  (Otto C. Watzka and Co.) i n x y l e n e .  graphed under h i g h power  The c o v e r  mounting  G-banded metaphases were p h o t o -  (lOOx o b j e c t i v e ) u s i n g a Z e i s s photomicroscope I I I  w i t h a u t o m a t i c l i g h t m e t e r i n g on Kodak h i g h c o n t r a s t copy f i l m .  F i l m s were  developed i n Kodak D19 d e v e l o p e r and p r i n t e d on grade 3 paper. The method used f o r producing.Q-bands Caspersson 20 minutes.  was a m o d i f i c a t i o n o f t h a t o f  (84). Cover g l a s s e s were immersed i n a t e b r i n  (Gurr 2900) f o r  The cover g l a s s e s were mounted on s t a n d a r d microscope  slides  i n a 5% s u c r o s e s o l u t i o n and s e a l e d around t h e edges w i t h rubber cement. Metaphases  were photographed u s i n g a Z e i s s photomicroscope I I I w i t h I V F l -  e p i - f l u o r e s c e n c e and a Z e i s s 487709 e x c i t e r / b a r r i e r f i l t e r c o m b i n a t i o n . Photographs were taken u s i n g spot m e t e r i n g on Kodak p l u s X f i l m pushed t o ASA 300, and d e v e l o p e d i n D i a f i n e d e v e l o p e r .  Photographs were p r i n t e d on  grade 4 paper w i t h the n e g a t i v e s l i g h t l y o u t o f f o c u s t o reduce g r a i n i n e s s .  5.  D i f f e r e n t i a l Staining of Sister  a.  Rationale S i s t e r c h r o m a t i d exchanges  of  Chromatids  a r e thought t o r e p r e s e n t the i n t e r c h a n g e  DNA r e p l i c a t i o n p r o d u c t s a t a p p a r e n t l y homologous chromosomal l o c i .  These exchanges the  presumably  i n v o l v e DNA  f r e q u e n c y o f such exchanges  d e t e c t DNA  and r e u n i o n .  A n a l y s i s of  p r o v i d e s a s e n s i t i v e c y t o l o g i c a l method to  i n t e r c h a n g e s f o l l o w i n g DNA  question the v a l i d i t y  breakage  damage ( 8 5 ) .  Some a u t h o r s ( e . g . Ref. 2)  o f k a r y o t y p e s o b t a i n e d from c e l l s  t h a t have undergone  more than a few d i v i s i o n s i n c u l t u r e because o f the u n d e f i n e d r o l e o f potent i a l in vitro  agents t h a t may  possible clastogenic the of  c u l t u r e o f the h e m o p o i e t i c c e l l s sister  c h r o m a t i d exchanges  used throughout t h i s work, the frequency  i n such c e l l s was  grown in vitro  d e t e r m i n e d and compared t o  f o r much s h o r t e r p e r i o d s o f t i m e .  Technique i.  Bone Marrow  0.1  ml o f f r e s h bone marrow a s p i r a t e was  (Connaught of  i n c u b a t e d i n 5 ml o f a-medium  L a b o r a t o r i e s 1403-01-07) w i t h 20% f e t a l  e r y t h r o p o i e t i n p e r ml  c a l f serum and 0.25  i n c u b a t i o n a t 37°C i n a 5% CO^ added  s i o n was at  i n a i r environment,  10  3  t o g i v e a f i n a l c o n c e n t r a t i o n o f 50 yM 5BrdU.  i n the dark u n t i l  units  (epo, Step I I I , Connaught L a b o r a t o r i e s ) i n a s t e r i l e  15 ml g l a s s t e s t tube w i t h a l o o s e l y f i t t i n g screw cap.  was  In o r d e r t o e v a l u a t e  ( i . e . chromosome damaging) e f f e c t s o f the system used i n  t h a t o b t a i n e d from c e l l s  b.  cause chromosomal damage.  t h e time o f h a r v e s t .  A f t e r e i g h t hours o f M 5BrdU (Sigma B-5002) C u l t u r e s were kept  One hour p r i o r t o h a r v e s t , c e l l d i v i -  a r r e s t e d a t metaphase by the a d d i t i o n o f c o l c e m i d (Gibco #120-5210)  a f i n a l c o n c e n t r a t i o n o f 0.1 yg/ml.  a f t e r the a d d i t i o n o f 5BrdU.  C u l t u r e s were h a r v e s t e d 24 t o 91 hours  At the end o f t h i s i n c u b a t i o n p e r i o d , the  cells  were g e n t l y resuspended i n the growth tubes and then emptied i n t o c o v e r e d c e n t r i f u g e tubes and spun down a t 800 rpm of  the s u p e r n a t a n t was  5 ml o f h y p o t o n i c KC1  discarded. (0.075 M).  were c e n t r i f u g e d a t 800 rpm  The  f o r e i g h t minutes.  c e l l s were c a r e f u l l y  A l l b u t 0.05  ml  resuspended i n  A f t e r s t a n d i n g f o r 20 minutes, the  cells  f o r e i g h t minutes, and a g a i n , a l l b u t a few drops  -30-  of  the s u p e r n a t a n t was  discarded.  The c e l l s were resuspended i n 5 ml o f c o l d methyl a l c o h o l : g l a c i a l a c e t i c a c i d ) . first  amount o f f i x a t i v e was  added  (3:1 a b s o l u t e  To reduce clumping o f the c e l l s ,  The remainder o f t h e f i x a t i v e  The tubes were a l l o w e d t o s t a n d a t room temperature  The c e l l s were then c e n t r i f u g e d a t 800 rpm supernatant was  discarded.  f i x a t i v e a f t e r the i n i t i a l  was  f o r 20 m i n u t e s .  f o r e i g h t minutes  The c e l l s were washed two  and the  t o t h r e e times i n c o l d  fixation.  A f t e r the f i n a l wash, t h e s u p e r n a t a n t was  removed and the c e l l s were  resuspended i n f i v e o r s i x drops o f f r e s h f i x a t i v e .  About t h r e e drops o f  t h i s s u s p e n s i o n were dropped onto t h r e e drops o f a 30% g l a c i a l a c e t i c s o l u t i o n on a p r e c l e a n e d s l i d e from a h e i g h t o f about 8 cm t o s p r e a d i n g o f the chromosomes. to  the  s l o w l y and the c e l l s were immediately  gently agitated with a Pasteur p i p e t t e . then added.  fixative  The s l i d e was  acid  facilitate  g e n t l y blown on and then a l l o w e d  a i r dry o v e r n i g h t .  ii.  Erythropoietic Colonies  To o b t a i n d i f f e r e n t i a l s t a i n i n g o f s i s t e r c h r o m a t i d s from chromosomes o b t a i n e d from e r y t h r o i d c o l o n i e s , of the  growth and a t a f i n a l  5BrdU were added  c o n c e n t r a t i o n o f 50 yM.  t o each c u l t u r e on Day F o r t y - e i g h t hours  9  after  a d d i t i o n o f 5BrdU, the c u l t u r e s were h a r v e s t e d as d e s c r i b e d i n s e c t i o n  iii.  Differential  After at l e a s t were immersed i n a 0.5  Staining  24 hours o f a i r d r y i n g , the s l i d e s o r c o v e r g l a s s e s ug/ml aqueous s o l u t i o n o f B i s b e n z i m i d Hoechst  (Riedel-De Haen Ag S e e l z e Hannover #33217) f o r 13 minutes i n water.  The f l u o r e s c e n c e p l u s Giemsa t e c h n i q u e used was  associates  (86), w i t h t h e f o l l o w i n g m o d i f i c a t i o n s :  33258  and then r i n s e d t h a t o f Goto  and  s l i d e s or cover glasses  3.  -31-  s t a i n e d w i t h Hoechst about  33258 were p l a c e d i n p e t r i  2 cm o f phosphate  0.04 M sodium  citrate).  c i t r a t e b u f f e r pH 7.0 The p e t r i  d i s h e s and c o v e r e d w i t h  (0.16 M sodium  d i s h e s were t h e n c o v e r e d t o minimize  e v a p o r a t i o n , and the d i s h and i t s c o n t e n t s exposed light  t o 24 hours o f c o n t i n u o u s  from a 150 watt grow lamp ( S y l v a n i a s p o t - g r o w ) .  a t a d i s t a n c e o f 35 cm from the l i g h t s o u r c e . slides  phosphate-  The d i s h e s were k e p t  A f t e r l i g h t exposure, the  o r c o v e r g l a s s e s were r i n s e d b r i e f l y i n d i s t i l l e d water  s t a i n e d i n 5% Giemsa ( H a r l e c o No. 620) i n 2:3 phosphate d i s t i l l e d water,  f o r eight  and then  b u f f e r pH  6.8:  minutes.  Cover g l a s s e s were mounted i n 25% E u k i t t mounting media i n x y l e n e , and metaphases w i t h chromosomes showing chromatids were photographed,  differential  staining of sister  u s i n g a Z e i s s photomicroscope  m a t i c l i g h t m e t e r i n g , on Kodak h i g h c o n t r a s t copy  film.  I I I w i t h auto-  F i l m s were  d e v e l o p e d i n Kodak D19 d e v e l o p e r and p r i n t e d on grade 4 p a p e r . of  sister  graphic  Frequencies  c h r o m a t i d exchanges were determined by t h e a n a l y s i s o f t h e photo-  prints. For  c e l l c y c l e k i n e t i c s e x p e r i m e n t s , number o f c e l l s i n f i r s t ,  second, and t h i r d d i v i s i o n  metaphase were s c o r e d  visually.  -32-  RESULTS  1.  Time Course S t u d i e s  a.  Cell  Recovery  B e f o r e d e t e r m i n i n g t h e day(s) when maximal numbers o f metaphases c o u l d be 'obtained from e r y t h r o i d c o l o n i e s , time c o u r s e t o t a l r e c o v e r a b l e number o f c e l l s  studies o f the  from i n d i v i d u a l c o l o n i e s were r u n .  C o l o n i e s were h a r v e s t e d a s i f f o r chromosome a n a l y s i s and t o t a l counts performed  on each cover g l a s s .  cell  On Day 8 o f c u l t u r e , t h e s i x  l a r g e s t e r y t h r o i d c o l o n i e s t h a t c o u l d be found on f o u r c u l t u r e d i s h e s were p l u c k e d and h a r v e s t e d i n d e p e n d e n t l y .  T h i s procedure  every day, u s i n g d i f f e r e n t c u l t u r e d i s h e s , f o r t h e next  was r e p e a t e d seven days.  The  c o l o n i e s . s e l e c t e d were v i s u a l l y e s t i m a t e d as c o n t a i n i n g s e v e r a l hundred t o a few thousand  cells.  Two s e p a r a t e experiments  d i f f e r e n t p e r i p h e r a l b l o o d .samples. F i g u r e 3a,b.  using  The r e s u l t s a r e shown g r a p h i c a l l y i n  A maximum r e c o v e r y o f a p p r o x i m a t e l y  obtained a f t e r  were performed  500 c e l l s c o u l d be  11 t o 12 days i n c u b a t i o n .  The d a t a agree w i t h p r e v i o u s o b s e r v a t i o n s on t h e growth k i n e t i c s of erythroid colonies,  i . e . , most o f t h e l a r g e s t e r y t h r o i d c o l o n i e s r e a c h  t h e i r maximum s i z e by two weeks. have reached  By t h i s time, v i r t u a l l y a l l o f t h e c e l l s  t h e n o n d i v i d i n g e r y t h r o b l a s t stage o f m a t u r a t i o n  and soon  a f t e r , the c e l l s e x h i b i t the f r a g i l e c h a r a c t e r i s t i c s o f maturing r e d cells  (72). The p r e s e n t r e s u l t s suggest t h a t some c o l o n y  disintegration  b e g i n s around Day 11 o r 12 i n many normal e r y t h r o i d c o l o n i e s and thus maximum numbers o f metaphases would n o t u s u a l l y be a n t i c i p a t e d beyond Day 12.  -33-  _L  1  t  r  w  Number of Days in Culture Before Colony Harvest  F i g u r e 3a.  T o t a l r e c o v e r y o f c e l l s from a s i n g l e c o l o n y a f t e r h y p o t o n i c treatment, f i x a t i o n , and two washes, shown as a f u n c t i o n o f t h e day o f c o l o n y h a r v e s t . Each p o i n t r e p r e s e n t s t h e mean r e c o v e r y o f s i x c o l o n i e s h a r v e s t e d independently. Bars i n d i c a t e the standard e r r o r . C u l t u r e s i n i t i a t e d from p e r i p h e r a l b l o o d c e l l s from ID.  -34-  F i g u r e 3b.  T o t a l r e c o v e r y o f c e l l s from a s i n g l e c o l o n y a f t e r h y p o t o n i c treatment, f i x a t i o n , and two washes, shown as a f u n c t i o n o f the' day o f c o l o n y h a r v e s t . Each p o i n t r e p r e s e n t s the mean r e c o v e r y o f s i x c o l o n i e s h a r v e s t e d independently. B a r s i n d i c a t e the s t a n d a r d e r r o r . C u l t u r e s i n i t i a t e d from p e r i p h e r a l b l o o d c e l l s from KH.  -35-  b.  M i t o t i c Index  Previous  s t u d i e s on the growth c h a r a c t e r i s t i c s o f e r y t h r o p o i e t i c  c o l o n i e s have suggested t h a t p r o l i f e r a t i o n and associated  (66).  Thus, most c e l l s i n a g i v e n  m a t u r a t i o n are c l o s e l y colony  s t a t e s o f d i f f e r e n t i a t i o n a t a p p r o x i m a t e l y the the p r o d u c t i o n after, and,  hence, e a s i e r t o r e c o g n i z e , I t was  begin to f a l l  i n the  but  the  final  At t h i s  point,  living cells.  There-  become much r e d d e r  c o l o n i e s do not  increase  further  a n t i c i p a t e d , t h e r e f o r e , t h a t the m i t o t i c index would  dramatically  j u s t before  c o l o n i e s r e a c h e d t h e i r maximum  E x p e r i m e n t s were then undertaken t o d e t e r m i n e the time dependence  of m i t o t i c index. using  same t i m e .  the c e l l s c o n t i n u e t o produce hemoglobin and  in size.  size.  o f hemoglobin becomes v i s i b l e  r e a c h the  T h i s was  done f i r s t u s i n g p o o l e d c o l o n i e s and  then  single colonies.  i.  Pooled  Colonies  F i v e c u l t u r e s from d i f f e r e n t donors were s e t up colonies. found on  On  Day  10,  the  six l a r g e s t e r y t h r o i d colonies that could  four c u l t u r e dishes  chromosome h a r v e s t  were p o o l e d b e f o r e  procedure.  The  repeated every  f o r the n e x t n i n e days.  per  c o v e r g l a s s , were  Following  routine total  scored.  r e s u l t s , summarized i n T a b l e I I I , i n d i c a t e t h a t t h e r e was  c o n s i s t e n t change i n the m i t o t i c i n d e x on d i f f e r e n t days o f between Days 6 and  15,  when p o o l e d c o l o n i e s were used.  m i t o t i c i n d e x averaged about  1.5%.  the  day,  the t o t a l number o f metaphases, as w e l l as the  number o f c e l l s r e c o v e r e d  be  the h y p o t o n i c s t e p o f  T h i s p r o c e d u r e was  using d i f f e r e n t culture dishes, chromosome h a r v e s t ,  for erythroid  harvest,  Overall,  the  no  Table I I I .  Percentage  of Recovered C e l l s i n Metaphase i n Pooled Erythroid Colonies Harvested  On Different Days of Culture.  Specimen  — HI  ID  Six Colonies Were Pooled to Give Each Value.  Percentage of C e l l s i n Metaphase ^ ^ MD TS :  * PC  Mean  Day of Harvest 6  No recovery  0  0  1.23  0.46  0.4 ± 0.3  7  0.23  0  0  3.15  0  0.7. ±0.6  8  1.23  0.79  0.50  1.88  1.76  1.2 ± 0.3  9  0.87  0  ' 0.12  3.02  3.06  1.4 ±0.7  10  3.99  6.70  1.23  1.07  2.82  3.2 ±1.0  11  1.07  1.03  1.25  0.33  1.05  1.0 ±0.2  12  1.59  0.16 .  0.60  0.87  1.00  0.8 ± 0.2  13  0.71  0.25 '  0.75  6.56  0.63  1.8 ±1.2  14  No recovery  0.92  2.41  0  0.21  0.9 ± 0.6  15  No recovery  0.71  0.71  0  0.98  0.6 ± 0.2  16  No recovery  No recovery  No recovery  0  No recovery  0  Mean  1.4 ±0.5  1.0±0.7  0.7±0.2  1.7±0.6  1.2±0.4  Overall Mean 1.2 ± 0. 3  *  Colonies i n i t i a t e d from peripheral blood. **  "  -  v  Colonies i n i t i a t e d from bone marrow.  -37-  The p o s s i b i l i t y t h a t w i t h i n each c o l o n y the c e l l s were s y n c h r o n i z e d , but t h a t d i f f e r e n t c o l o n i e s were a t d i f f e r e n t c e l l c y c l e a t any g i v e n time, c o u l d not be e x c l u d e d . examination  s t a g e s o f the  A more r i g o r o u s  o f p o s s i b l e i n t e r c o l o n y v a r i a t i o n s i n m i t o t i c index was  by r e p e a t i n g the experiment  ii.  using single erythroid  Single Colonies  f o r chromosomes on each day  Two  from Day  e s t i m a t e d as a p e r c e n t a g e  experiments  There was  harvested  9 t o Day.13 o f c u l t u r e .  The  mitotic  o f the r e c o v e r e d c e l l s i n metaphase.  were run u s i n g d i f f e r e n t  g r a p h i c a l l y i n F i g u r e s 4a and  specimens.  The d a t a are shown  4b.  a l a r g e v a r i a t i o n i n t h e m i t o t i c index f o r d i f f e r e n t  c o l o n i e s from the same i n d i v i d u a l h a r v e s t e d a t the same time. range on any g i v e n day was t h e r e was,  made  colonies.  Ten e r y t h r o i d c o l o n i e s were p l u c k e d i n d e p e n d e n t l y and  index was  relatively  from ,0 t o 6%  ( F i g . 4a, Day  10).  The  largest  However,  a g a i n , no c o n s i s t e n t t r e n d d u r i n g the time i n t e r v a l s t u d i e d .  The a b s o l u t e number o f metaphases r e c o v e r e d per c o l o n y i s a l s o shown i n F i g u r e s 4a and  4b.  These ranged  from z e r o t o f i v e , w i t h  s i o n a l c o l o n i e s y i e l d i n g up t o 21 metaphases. were o b t a i n e d from 9- t o 11-day-old colonies.  On  average,  more metaphases  c o l o n i e s than from 12- t o  In most c a s e s , the number o f metaphases p e r c o l o n y  the m i t o t i c indices..  occa-  13-day-old parallels  T h i s i n d i c a t e s t h a t m i t o t i c c e l l s were not  prefer-  e n t i a l l y gained or l o s t during the h a r v e s t i n g procedure. The suggested  s i n g l e c o l o n y d a t a , s i m i l a r t o those f o r the p o o l e d the average  colonies,  m i t o t i c index does not v a r y markedly w i t h the day  h a r v e s t between Days 9 and  13.  of  However, marked i n t e r c o l o n y v a r i a t i o n does  -38-  Day of Colony Harvest  j»  11  10  12  13  ihas  20  |  r-  15  o  NumbeIT of Cells  c  •  •  0  'IT  9  M  10  rllff  11  12  ir  •  -  r l f l l 13  F  F  j-r  A.  F i g u r e 4a.  rf  1 rf : nrrr Colon I M  1  .rfflf  Percentage o f r e c o v e r e d c e l l s i n metaphase. Ten c o l o n i e s were p l u c k e d on each day f o r Days 9 t o 13 o f i n c u b a t i o n . A l s o shown i s t h e a b s o l u t e number o f metaphases o b s e r v e d per corresponding colony. The average number o f c e l l s r e c o v e r e d p e r c o l o n y was 268 ± 1 6 9 . The range was from 27 t o 752. C u l t u r e s i n i t i a t e d from bone marrow c e l l s from A J .  -39-  Day ol Colony 9  20  10  Harvest  11  12  J3  cn CO  of Cells in Metap  SZ  5  JD  E z  - F-fh-n  rf  0  10  9  rrfTrrr n r r r13. i;>  5 0) cn ro  SZ Q.  £  0)  4  -  5  c  Percentage of Ci  «  -r pf!  rf  j-T  rf"  rftl  nTff  Colonial  F i g u r e 4b.  P e r c e n t a g e o f r e c o v e r e d c e l l s i n metaphase. Ten c o l o n i e s were p l u c k e d on each day f o r Days 9 t o 13 o f i n c u b a t i o n . A l s o shown a r e the a b s o l u t e number o f metaphases o b s e r v e d per c o l o n y . C u l t u r e s i n i t i a t e d from bone marrow c e l l s from HS.  -40-  o c c u r and i s p r e s e n t throughout  t h i s period.  In subsequent  c o l o n i e s were h a r v e s t e d between Days 9.and 11.  Colony s i z e and c o l o u r  were a l s o c o n s i d e r e d when e s t i m a t i n g the b e s t day  .2.  S i s t e r Chromatid  experiments,  f o r chromosome h a r v e s t .  Exchange S t u d i e s  The c o m p l e x i t y o f k a r y o t y p i c changes i n some human c e l l s grown i n long-term c u l t u r e s has g i v e n some a u t h o r s r e a s o n t o e x c l u d e such d a t a consideration  (2).  The  s i s t e r c h r o m a t i d exchange  (SCE)  assay i s considered  t o be much more s e n s i t i v e t o c l a s t o g e n s ( i . e . agents t h a t cause breakage) t h a n the chromosomal a b e r r a t i o n assay  (87).  from  chromosomal  In o r d e r t o d e t e r -  mine whether the c u l t u r e c o n d i t i o n s used f o r the growth o f e r y t h r o i d c o l o n i e s were c l a s t o g e n i c , c o l o n i e s was  the SCE  f r e q u e n c y i n 1- to' 2-week-old e r y t h r o i d  compared t o t h a t o f e r y t h r o p o i e t i n - s t i m u l a t e d  f r e s h marrow  cells. The t e c h n i q u e used t o produce  differential  staining of  sister  chromatids e n t a i l s two  rounds of. DNA  base analogue,  T h i s time, p e r i o d i s a p p r o x i m a t e l y 24 hours f o r  5BrdU.  d i v i d i n g lymphocytes  (88), 48 hours  r e p l i c a t i o n i n the p r e s e n c e o f the  for dividing fibroblasts  48 hours f o r f r e s h marrow p r e p a r a t i o n s (90).  There i s no p u b l i s h e d d a t a  on t h i s a s p e c t o f e r y t h r o i d c o l o n y growth k i n e t i c s . e s t i m a t e o f t h e time r e q u i r e d  (89), and  To get an  approximate  f o r e r y t h r o p o i e t i c c e l l s t o p r o g r e s s from  s y n t h e s i s t o metaphase o f the second c y c l e , a time c o u r s e experiment run u s i n g f r e s h marrow c e l l s .  I n o r d e r t o enhance the p r o p o r t i o n o f  e r y t h r o i d c e l l s i n the d i v i d i n g p o p u l a t i o n a n a l y z e d , e r y t h r o p o i e t i n added.  In the absence  was  o f t h i s hormone, the v i a b i l i t y o f e r y t h r o i d  r a p i d l y decreases i n short-term c u l t u r e  (91).  was cells  -41-  a.  F r e s h Marrow  M y e l o i d c e l l s were c u l t u r e d  from 24 t o 91 hours i n t h e p r e s e n c e o f  5BrdU,. and then h a r v e s t e d f o r chromosomes and s t a i n e d f o r d i f f e r e n t i a l l a b e l l i n g of s i s t e r chromatids.  One  thousand c e l l s were a n a l y z e d f o r each o f  f i v e , independent h a r v e s t s and t h e number o f c e l l s  in first,  t h i r d d i v i s i o n metaphase were s c o r e d .  were i d e n t i f i e d as b e i n g  either f i r s t ,  second, o r t h i r d d i v i s i o n by the d i s t r i b u t i o n o f 5BrdU,  v i s u a l l y d i s c e r n a b l e as t h e l i g h t e r (see  Metaphases  second, and  F i g . 5).  s t a i n i n g r e g i o n s , i n the c h r o m a t i d  D u p l i c a t e e x p e r i m e n t s were r u n u s i n g two d i f f e r e n t  specimens.  The r e s u l t s a r e shown g r a p h i c a l l y i n F i g u r e s 6a and 6b, and c o n f i r m the  that  maximum number o f second d i v i s i o n metaphases c o u l d be o b t a i n e d by  i n c u b a t i n g f r e s h marrow f o r 48 h o u r s .  b.  Erythroid  Colonies  The i n f o r m a t i o n g a i n e d by the p r e v i o u s experiment was differential  s t a i n i n g o f s i s t e r c h r o m a t i d s i n metaphases from p o o l e d  erythroid colonies. For  used t o o b t a i n  F o r t y - e i g h t hours p r i o r t o h a r v e s t , 5BrdU was  added.  each o f f o u r specimens, groups o f s i x t o 12 e r y t h r o i d c o l o n i e s  p o o l e d p r i o r t o the h y p o t o n i c s t e p were p r o c e s s e d .  A f t e r r o u t i n e chromo-  some h a r v e s t , and s t a i n i n g p r o c e d u r e , metaphases showing s t a i n i n g chromatids were o b s e r v e d r e g u l a r l y .  One  l e a s t 30 d i f f e r e n t i a l l y s t a i n e d metaphases was and s c o r e d f o r s i s t e r c h r o m a t i d exchanges. ranged from 3.1  t o 5.3 p e r c e l l .  ;  differentially  coverglass containing at  s e l e c t e d f o r each  specimen  The mean f r e q u e n c y o f SCE  The r e s u l t s a r e shown i n T a b l e IV.  These v a l u e s f a l l w i t h i n t h e range o b s e r v e d f o r f r e s h marrow t r e a t e d i n the  same way.  (The v a l u e shown i n T a b l e IV i s t a k e n from t h e d a t a i n  -42-  F i g u r e 5.  C e l l c y c l e k i n e t i c s e v a l u a t i o n u s i n g 5BrdU i n c o r p o r a t i o n and t h e . d i f f e r e n t i a l s t a i n i n g t e c h n i q u e . Metaphases a f t e r (a) one, (b) two, and (c) t h r e e s y n t h e s e s i n 5 BrdU. A l l t h r e e metaphases were t a k e n from one bone marrow c u l t u r e from an i n d i v i d u a l w i t h Bloom syndrome.  -43-  -44-  80  70  60 2nd Division Metaphases 50H co  O  CD XI  40 1st Division Metaphases  E  1  \  30&\  \  3rd Division Metaphases  20  10  Al T  T  i.  60 80 40 20 Incubation Period in 5 BrdU (Hours)  F i g u r e 6a.  100  The number o f c e l l s i n f i r s t , second, and t h i r d metaphase . a f t e r v a r i o u s l e n g t h s o f time i n i n c u b a t i o n i n 50 ]XM 5BrdU. One thousand c e l l s were s c o r e d a t each h a r v e s t . Bars i n d i c a t e the standard e r r o r . C u l t u r e s i n i t i a t e d from bone marrow c e l l s from NF.  -45-  80  70  60*1st Division Metaphases 50 CD  o 40 0)  E Z  30  ,  T  2nd Di vision M e t a p h a s e s  20  T ' •V  v  10  J\ /  5 ?  MS  40  \ /  •  *I  60  3rd Division Metaphases  » ^'*»*  Incubation Period in 5 BrdU  F i g u r e 6b.  \/  80  :  1 0 0  (Hours)  The number o f c e l l s i n f i r s t , second, and t h i r d metaphase a f t e r v a r i o u s l e n g t h s o f time i n i n c u b a t i o n i n 50 yM 5BrdU. One thousand c e l l s were s c o r e d a t each h a r v e s t . Bars i n d i c a t e the s t a n d a r d e r r o r . C u l t u r e s i n i t i a t e d from bone marrow c e l l s from HS.  Table IV.  Experiment „ No.  Average Number of S i s t e r Chromatid •Four Individuals. For comparison, normal f i b r o b l a s t s , normal marrow, Bloom syndrome. In a l l cases, the ^. . Diagnosis ^  Exchanges Per C e l l i n Pooled Erythroid Colonies From the SCE frequency i s shown f o r normal.lymphocytes, and lymphocytes and marrow from an individual with f i n a l concentration of 5BrdU was 50 uM. „ C e l l Type n n  .  m  No. C e l l s ^ . Counted  SCE/Metaphase , „„ ± SE  Myeloma  Erythroid colonies from marrow  30  3.6±0.7  WK  Erythrocytosis  Erythroid colonies from marrow  30  4.8 ± 0.5  DL  Myeloma  Erythroid colonies from marrow  30  3.1±0.6  GK  Normal  Erythroid colonies from peripheral blood  30  5.3 ± 0.5  HS  Normal  Epo-stimulated fresh marrow  30  3.810.4  Normal  Epo-stimulated fresh marrow  15  4.5 1 0.6  NN HT12 HT21 HT31  i i i  HT41* * FT1  Normal  PHA-stimulated  peripheral blood  30  3.110.4  Normal  PHA-stimulated  peripheral blood  50  4.6 1 0.3  Normal  PHA-stimulated  peripheral blood  46  3.910.3  Normal  PHA-stimulated  peripheral blood  79  3.7 1 0.2  Skin fibroblasts-passage s i x  30  4.210.4  Normal  . JB  Bloom syndrome  JB  Bloom syndrome  Epo-stimulated fresh marrow PHA-stimulated  *  Values taken from a previous study (92).  peripheral blood  5  115 126  10  97 1 22  -47-  s e c t i o n 2a.)  F o r comparison,  the SCE  c y t e s i s shown f o r f o u r i n d i v i d u a l s .  frequency  i n normal human lympho-  (These v a l u e s were o b t a i n e d i n a  p r e v i o u s study i n which the same s t a i n i n g c o n d i t i o n s were used SCE  frequency determined  human s k i n i s a l s o g i v e n . PHA  i n a sample o f c u l t u r e d f i b r o b l a s t s from To o b t a i n a p o s i t i v e c o n t r o l ,  stimulated p e r i p h e r a l blood c e l l s  t h i s d i s e a s e i s shown i n T a b l e These r e s u l t s suggest  e r y t h r o i d c e l l s produced tically  3.  normal  f r e s h marrow and  from a p a t i e n t w i t h Bloom syndrome  were a n a l y z e d , and the g r e a t l y enhanced f r e q u e n c y o f SCE of  (92)).  characteristic  IV.  t h a t under the c u l t u r e c o n d i t i o n s used,  a f t e r - o n e to. two  weeks..incubation  are  the  cytogene-  stable.  P r o o f o f the S i n g l e C e l l  O r i g i n o f I n d i v i d u a l l y Plucked  Colonies  In o r d e r t o show t h a t a s i n g l e c o l o n y o f c e l l s c o n t a i n i n g about s e v e r a l hundred c e l l s c o u l d be p l u c k e d  from a 35 mm  d i s h containing at  l e a s t 30 s i m i l a r c o l o n i e s , the f o l l o w i n g m i x i n g experiment P e r i p h e r a l b l o o d samples were t a k e n v o l u n t e e r and p r e p a r e d two.  samples  Fifty-six to  were  i n the u s u a l manner.  mixed  from t h e s e ,  These were then Q-banded and  female  E q u a l numbers- o f c e l l s from t h e dish.  f o r chromosomes on Days 11  32 p r e p a r a t i o n s y i e l d e d metaphases.  s c o r e d f o r the number o f c e l l s t h a t c o u l d  be p o s i t i v e l y i d e n t i f i e d as male o r female. l a r g e l y due  from a male and a  performed.  and p l a t e d a t 1 x 10^ c e l l s o f each type p e r  e r y t h r o i d c o l o n i e s were h a r v e s t e d  13 o f growth, and  was  I n c o n c l u s i v e metaphases,  to- poor s p r e a d i n g o f the metaphases o r m i s s i n g chromosomes,  were a l s o r e c o r d e d .  The  r e s u l t s a r e shown i n T a b l e V.  Of 32  individual  c o l o n i e s i n which metaphases were o b t a i n e d , 16 were i d e n t i f i e d as male and  -48-  T a b l e V.  D i s t r i b u t i o n o f F l u o r e s c e n t Y-Body i n I n d i v i d u a l C o l o n i e s P l u c k e d From D i s h e s i n Which E q u a l Numbers o f Male and Female C e l l s Were P l a t e d . Number o f Metaphases  Colony  Conclusion Y-Body P o s i t i v e  Y-Body N e g a t i v e  1 2 3 4 5 6 7 8 9 10 11 12 13 14  0 0 0 0 0 0 0 0 0 0 0 0 0 0  24 3 38 1 4 7 8 49 18 1 8 17 6 24  15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30  3 15 8 34 31 10 4 13 61 49 26 11 11 34 22 1  31 32  0 0  Inconclusive  . 1 6 12 20 3 1 13 12 21 3 4 18 4 12  Female Female Female Female Female Female Female Female Female Female Female Female Female Female  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 3 4 10 2 4 2 8 18 2 1 0 0 2 1 1  Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male  0 0  2 2  Inconclusive Inconclusive  1  -49-  14 as female.  In no i n s t a n c e  i n the c e l l s r e c o v e r e d  were b o t h male and female metaphases found  from a s i n g l e c o l o n y .  Two c o l o n i e s y i e l d e d meta-  phases t h a t gave i n c o n c l u s i v e r e s u l t s .  4.  K a r y o t y p e s From S i n g l e  Extensive  chromosome  Colonies  a n a l y s i s was performed on s i n g l e e r y t h r o i d  c o l o n i e s from s i x i n d i v i d u a l s .  I n each case, t e n c o l o n i e s were s e l e c t e d  on t h e b a s i s o f s i z e and c o l o u r . Days 9 and 14 o f i n c u b a t i o n . under the m i c r o s c o p e .  A l l c o l o n i e s were h a r v e s t e d  Metaphases were G- o r Q-banded and  metaphases o b t a i n e d  T a b l e VI shows t h e average number o f  per colony  i n each c a s e .  the q u a l i t y o f G- and Q-banded k a r y o t y p e s o b t a i n a b l e colonies.  analyzed  A t l e a s t one G-banded and one Q-banded k a r y o t y p e  were p r e p a r e d f o r each i n d i v i d u a l . analyzable  between  Figures  7-10 show  from s i n g l e e r y t h r o i d  -50-  Table VI.  „ ,. ^ Sub ect D  Average Number o f A n a l y z a b l e Metaphases Per C o l o n y f o r Six Individuals. Ten C o l o n i e s Were P l u c k e d i n Each Case and Q- o r G-banded. Number o f Metaphases p e r Colony (Ave.iSE)  Karyotype  WH  3.6±1.0  46, XY  RA2  4.211.1  46, XY  EM  5.4 ± 0.9  46, XX  KS  3.3 ± 1 . 1  46, XY  PD  6.4 ± 1.2  46, XY  WS  3.9 ± 0.7  46, XY  -51-  %  >  /  «  J: i l ((  II II )>  a if ^ i\ II ii *  11  1  n  F i g u r e 7.  14  J?  15  »  It H  ii  i 17  II  l  l 11  JO  Q-banded male k a r y o t y p e o b t a i n e d from a s i n g l e e r y t h r o i d colony grown from p e r i p h e r a l b l o o d c e l l s from WH.  -52-  I i t  ii  1 II ii  i.  I  »  I  it n  14  it  JO  4  it  7  ii  II  15  i  it  10  ii  11  .1  1  5  •  i  It  17  >i  a  it  ii X  X  •  F i g u r e 8.  Q-banded female k a r y o t y p e o b t a i n e d from a s i n g l e c o l o n y  grown  from  bone  marrow  c e l l s  from EM.  erythroid  -53-  II -  T  •  il  A  h  n  ••  41  11  i7  u•1  ri  7  12  | fl  ii  14  *  0  22  F i g u r e 9.  G-banded male karyotype o b t a i n e d from a s i n g l e  colony grown from bone marrow c e l l s from WS.  t.  erythroid  -54-  Figure 1 0 .  G-banded female karyotype obtained from a single erythroid colony grown from peripheral blood c e l l s from EM.  -55-  DISCUSSION  There are o n l y a few  reports  i n the  a n a l y s i s of s i n g l e hemopoietic c o l o n i e s  l i t e r a t u r e of  (93,94,95,96).  i n c l u d e a n a l y s i s o f banded chromosomes, and been  i n no  None o f  instance  has  these a karyotype  published. In the p r e s e n t  study, a t e c h n i q u e has  h i g h q u a l i t y chromosomes ( s u i t a b l e f o r G-  been developed t h a t  o r Q-banding) t o be  from s i n g l e e r y t h r o i d c o l o n i e s grown from human b l o o d V a r i a b l e s t h a t a f f e c t the y i e l d o f a n a l y z a b l e sented.  For  the  somal b r e a k i n g  The  first  time, the  agents has  methylcellulose  c u l t u r e s f o r one  o r bone marrow.  metaphases have been  t o two  (a) On  average, 9-  r e s u l t s with respect  e n t i a t e d c o l o n i e s •in vitro;  (c) R e s u l t s  c u l t u r e s were c o n s i s t e n t w i t h the  G- or Q-banding, were o b t a i n e d  cell  and  to obtain  evidence differ-  o f a n a l y s i s o f mixed male-female showed  removed by the p l u c k i n g p r o c e d u r e used;  s i x high q u a l i t y karyotypes, s u i t a b l e f o r  from s i n g l e e r y t h r o i d c o l o n i e s .  major d i r e c t i o n s i n which the  recovery  showed no  analyz-  s i n g l e c e l l o r i g i n o f c o l o n i e s and  t h a t s i n g l e c o l o n i e s were, i n f a c t , average, between f o u r and  t o 11-day-old  to y i e l d of  o f a c l a s t o g e n i c e f f e c t throughout the p e r i o d r e q u i r e d  refined:  pre-  weeks.  (b) S i s t e r c h r o m a t i d exchange s t u d i e s  There are two  obtained  s i s t e r c h r o m a t i d exchange t e s t f o r chromo-  major f i n d i n g s are as f o l l o w s :  a b l e metaphases;  enables  been a p p l i e d t o e r y t h r o i d c o l o n i e s growing i n  e r y t h r o i d c o l o n i e s gave the b e s t  (d) On  cytogenetic  m i t o t i c index.  t e c h n i q u e may  Preliminary  have i n d i c a t e d t h a t the problem o f c e l l r e c o v e r y  may  be  be  further  experiments t o t a l l y overcome  by u t i l i z i n g t h e p o l y l y s i n e t r e a t m e n t t e c h n i q u e o f Rajendra  (97).  This  simple t e c h n i q u e i n v o l v e s the p r e t r e a t m e n t o f m i c r o s c o p e s l i d e s w i t h  -56-  the c a t i o n i c p o l y l y s i n e dispersed  (Sigma, P1886).  The  e n t i r e colony  i n t o a drop o f h y p o t o n i c s o l u t i o n on  the  can  treated slide.  negatively  charged c e l l s adhere t o the p o l y l y s i n e c o a t i n g , and  end  treatment p e r i o d ,  o f the  the c e l l s has  been  f i x e d immediately.  the h y p o t o n i c s o l u t i o n may  be  The the  removed  U s i n g t h i s method, c l o s e to 100%.  the m i t o t i c index by  ventional c e l l cycle blocking  the a d d i t i o n and  and  recovery  cannot be  removal o f  agents, such as m e t h o t r e x a t e  f e a s i b l e s i n c e an agent, once added t o the  s e m i s o l i d c u l t u r e medium,  removed w i t h o u t d i s r u p t i n g the c o l o n i e s .  t h y m i d i n e and  E x p e r i m e n t s i n which  but p r o v e d not  t o be  o f c u l t u r e s by c o l d shock  the c o l o n i e s are no  longer  • Recent.results  upon c o o l i n g .  has  has  a l s o been  accumulating m i t o t i c c e l l s  conditions,  entities.  g i v e b e t t e r r e s u l t s than c o l c e m i d (100).  Whether o r not  u s e f u l i n i n c r e a s i n g the y i e l d o f a n a l y z a b l e established.  tried  the p e c u l i a r  Under such  i d e n t i f i a b l e as d i s t i n c t  so f a r .  suggest t h a t the r e v e r s i b l e m i c r o t u b u l e  ( A l d r i c h ) , may  remains t o be  (99)  f e a s i b l e since methylcellulose  o f becoming less, v i s c o u s  nocodazole  excess  d i h y d r o f o l a t e r e d u c t a s e have been u n s u c c e s s f u l  Synchronization  con-  (98), i s not  attempts t o r e l e a s e methotrexate b l o c k s by the a d d i t i o n o f  colony  at  be  obtained.  Increasing  property  then  inhibitor,  i n terms o f  t h i s agent w i l l  be  metaphases from a s i n g l e  -57-  CONCLUSION  A m i c r o t e c h n i q u e has been d e v e l o p e d t h a t p e r m i t s the r o u t i n e c y t o g e n e t i c a n a l y s i s o f s i n g l e h e m o p o i e t i c c o l o n i e s grown i n s t a n d a r d m e t h y l cellulose cultures.  I t i s now  p o s s i b l e t o b e g i n a s y s t e m a t i c study o f  chromosomal involvement e x c l u s i v e l y i n the progeny  of individual  erythroid  stem c e l l s i n a s e r i e s o f newly d i a g n o s e d p a t i e n t s w i t h m y e l o p r o l i f e r a t i v e disease. U s i n g t h i s method, i t would be p o s s i b l e t o l o o k f o r normal  stem c e l l s i n P h ^ - p o s i t i v e CML,  chromosomally  and f o r s p e c i f i c marker chromosomes  i n stem c e l l s o f p a t i e n t s w i t h p o l y c y t h e m i a v e r a and e s s e n t i a l cytosis.  In p o l y c y t h e m i a v e r a , two  cytogenetically investigated:  thrombo-  t y p e s o f c o l o n y p o p u l a t i o n s may  be  those which have l o s t t h e i r dependence on  e r y t h r o p o i e t i n f o r growth, and t h o s e which r e q u i r e i t . The v a l u e o f the m i c r o t e c h n i q u e d e v e l o p e d here w i l l o n l y be  appre-  c i a t e d when the r e s u l t s . o b t a i n e d by i t s r o u t i n e use a r e compared w i t h the c y t o g e n e t i c p i c t u r e o b t a i n e d from d i r e c t bone marrow p r e p a r a t i o n s , and attempts are made t o c o r r e l a t e t h e s e chromosome f i n d i n g s w i t h the p i c t u r e , response t o t h e r a p y , d i s e a s e p r o g r e s s i o n , and p r o g n o s i s .  clinical  -58-  REFERENCES  1.  B o v e r i , T-. : Zur Frage der E n t s t e h u n g M a l i g n e r Tumoren. Gustav F i s c h e r , 1914, S. 1-64.  Jena,  2.  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K a k a t i , S., Abe, S., and Sandberg, A.A.: S i s t e r c h r o m a t i d exchange i n P h i l a d e l p h i a chromosome ( P h ) - p o s i t i v e leukemia. Cancer Res. 38, 2918-2921, 1978.  and Schreck, R.R.: S i s t e r c h r o m a t i d exchange a n a l y s i s . Genet. 32./ 297-313, 1980.  T r a n s f o r m a t i o n o f t h e s m a l l lymphocyte 1961.  i n culture.  1  91.  K r a n t z , S.B., and Jacobson, L.O.: E r y t h r o p o i e t i n and the R e g u l a t i o n of E r y t h r o p o i e s i s . Chicago, U n i v e r s i t y o f Chicago P r e s s , 1970.  92.  Dube, I.D.: The i n c i d e n c e o f s i s t e r c h r o m a t i d exchange i n c u l t u r e d human lymphocytes. A t h e s i s s u b m i t t e d i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r the degree o f B a c h e l o r o f S c i e n c e i n Zoology. The U n i v e r s i t y o f B r i t i s h Columbia, 1977, pp. 1-31.  -65-  93.  Aye, M.T., T i l l , J . E . , and M c C u l l o c h , E.A.: Cytological studies of g r a n u l o p o i e t i c c o l o n i e s from two p a t i e n t s w i t h c h r o n i c myelogenous leukemia. Exp. Hemat. 1, 115-118, 1973.  94.  C h e r v e n i c k , P.A., E l l i s , L.D., Pan, S.F., and Lawson, A.L.: Human leukemic c e l l s : In vitro growth o f c o l o n i e s c o n t a i n i n g the P h i l a d e l p h i a (Ph ) chromosome. S c i e n c e 174, 1134-1136, 1971. 1  95.  Moore, M.A.S., and M e t c a l f , D.: C y t o g e n e t i c a n a l y s i s o f human acute and c h r o n i c m y e l o i d leukemic c e l l s c l o n e d i n agar c u l t u r e . Int. J. Cancer 11, 143-152, 1973.  96.  B u l l , J . : C y t o g e n e t i c s t u d i e s o f marrow and p e r i p h e r a l b l o o d g r a n u l o c y t e c o l o n i e s i n t r e a t e d c h r o n i c myelogenous leukemia. B l o o d C e l l s 1^, 161-162, 1975.  97.  Rajendra,  98.  Y u n i s , J . J . , and C h a n d l e r , M.E.: H i g h - r e s o l u t i o n chromosome a n a l y s i s i n c l i n i c a l medicine. I n S t e f a n i n i , M., and H o s s a i n i , A. ( E d s . ) : P r o g r e s s i n C l i n i c a l P a t h o l o g y , V o l . V I I . New York, Grune and S t r a t t o n , 1977, pp. 267-288.  99.  Rjz5nne, M. , Vang N i e l s e n , K. , and Mogens, E. : E f f e c t o f c o n t r o l l e d c o l c e m i d exposure on human metaphase chromosome s t r u c t u r e . Hereditas 91, 49-52, 1979. . •-  100.  B.:  P e r s o n a l communication.  Z i e v e , G.W., T u r n b u l l , D., M u l l i n s , J.M., and M c i n t o s h , I.R.: P r o d u c t i o n o f l a r g e numbers o f m i t o t i c mammalian c e l l s by t h e use of the r e v e r s i b l e microtubule i n h i b i t o r hocodazole. Exp. C e l l . Res. 126, 397-405, 1980.  -66-  APPENDIX 1A  Procedure  f o r t h e P r e p a r a t i o n o f Hemopoietic  Stem C e l l s f o r P l a t i n g  From F r e s h Bone Marrow A s p i r a t e  A l l m a n i p u l a t i o n s o f human c e l l s were c a r r i e d out i n a l a m i n a r f l o w B i o g a r d hood under s t e r i l e c o n d i t i o n s . A l l apparatus which came i n t o d i r e c t c o n t a c t w i t h human c e l l s were a u t o c l a v e d b e f o r e they were discarded.  Procedure: 1)  Measure the t o t a l pipette.  volume o f marrow u s i n g a s t e r i l e  calibrated  2)  Remove 0.1 ml o f a s p i r a t e and use t h i s t o do a s t a r t i n g n u c l e a t e d c e l l count on the hemocytometer ( m a g n i f i c a t i o n : ' lOOx).  3)  U s i n g a s t e r i l e P a s t e u r p i p e t t e , t r a n s f e r the e n t i r e sample t o a 17 x 1 0 0 mm p l a s t i c tube ( F a l c o n 2057) and s p i n the specimen a t 1,000 rpm f o r 4 minutes a t room temperature (LEC HN-S centrifuge).  4)  Remove the b u f f y c o a t w i t h a s t e r i l e P a s t e u r p i p e t t e and t o another F a l c o n 2057 tube.  5)  L e t the tube s t a n d a t room temperature f o r 10-20 tate sedimentation of r e s i d u a l red c e l l s .  transfer  minutes t o  facili-  6) - T r a n s f e r the e n t i r e c o n t e n t s o f the tube, e x c e p t f o r the r e d b u t t o n , t o another F a l c o n 2057 tube, and add enough 2% f e t a l serum i n a-medium (2% FCS) t o b r i n g the volume up t o 10 ml. 7)  S p i n the specimen a t 950 rpm f o r 10 minutes a t 4°C automatic, r e f r i g e r a t e d c e n t r i f u g e ) .  8)  Pour o f f the s u p e r n a t a n t and resuspend of 10 ml.  9)  C e n t r i f u g e as i n s t e p 7.  i n 2% FCS  (Sorvall  cell calf  RC-3  t o g i v e a volume  10)  Pour o f f the s u p e r n a t a n t and r e s u s p e n d g i v e a f i n a l volume o f 3 ml.  11)  Remove 0.1 ml o f the c e l l s u s p e n s i o n and use t h i s t o do a c e l l count on the hemocytometer.  final  12)  D i l u t e the c e l l s  cells/ml).  i n 2% FCS  the specimen i n 2% FCS  ( f i n a l concentration of 2x 10  6  to  -67-  APPENDIX IB  Procedure  f o r t h e P r e p a r a t i o n o f Hemopoietic  From P e r i p h e r a l  Stem C e l l s  for Plating  Blood  A l l m a n i p u l a t i o n s o f human c e l l s were c a r r i e d o u t i n a l a m i n a r f l o w B i o g a r d hood under s t e r i l e c o n d i t i o n s . A l l a p p a r a t u s which came i n t o d i r e c t c o n t a c t w i t h human c e l l s were a u t o c l a v e d b e f o r e they were discarded.  Procedure: 1)  Measure t h e t o t a l volume o f b l o o d u s i n g a s t e r i l e pipette.  calibrated  2)  Remove 0.1 ml o f b l o o d and use t h i s t o do a s t a r t i n g n u c l e a t e d c e l l count on t h e hemocytometer ( m a g n i f i c a t i o n : lOOx).  3)  G e n t l y l a y e r 10 ml o f b l o o d on t o p o f 15 ml o f lymphocyte s e p a r a t i o n medium (Bionetics.8410-01) i n a s t e r i l e 50 ml c o n i c a l tube ( F a l c o n 2070).  4)  C e n t r i f u g e t h e specimen a t 1,700 rpm f o r 30 minutes a t room temperature (LEC HN-S c e n t r i f u g e ) .  5)  U s i n g a P a s t e u r p i p e t t e , c a r e f u l l y a s p i r a t e o f f and d i s c a r d t h e plasma l a y e r down t o 2 mm above t h e lymphocyte l a y e r .  6)  T r a n s f e r t h e lymphocyte l a y e r (3 t o 5 ml) t o a F a l c o n 2057 tube and add 2% FCS t o g i v e a f i n a l volume o f 10 m l .  7)  S p i n t h e specimen a t 950 rpm f o r 10 m i n u t e s a t 4°C ( S o r v a l l RC-3 automatic, r e f r i g e r a t e d c e n t r i f u g e ) .  8)  Pour o f f t h e s u p e r n a t a n t and resuspend  9)  S p i n as i n s t e p 7.  the p e l l e t  i n 10 ml 2% FCS.  the p e l l e t  i n 1 ml o f 2% FCS.  10)  Pour o f f t h e s u p e r n a t a n t and r e s u s p e n d  11)  Remove 0.1 ml o f t h e c e l l s u s p e n s i o n and use t h i s t o do a f i n a l , c e l l count on t h e hemocytometer.  12)  D i l u t e t h e c e l l s i n 2% FCS t o g i v e a f i n a l c o n c e n t r a t i o n o f 4 x 1 0 cells/ml.  6  -68-  APPENDIX 1C  Procedure  f o r the P r e p a r a t i o n o f t h e C u l t u r e Medium f o r Hemopoietic  Stem C e l l s  Medium was made up i n b a t c h e s o f 102 ml. Medium was f r o z e n i n 1 7 x 1 0 0 mm F a l c o n 2057 s t e r i l e d i s p o s a b l e p l a s t i c t u b e s . 2.7 ml o f medium was a l i q u o t e d per tube. F o r p l a t i n g , the tubes were thawed, and 0.3 ml o f the f i n a l c e l l s u s p e n s i o n was added t o each tube. After mixing.by v o r t e x , 1.1 ml o f the medium c o n t a i n i n g c e l l s were p l a t e d p e r 3 5 x 1 0 mm s t a n d a r d d i s h (Lux #5221-R) u s i n g p l a t i n g n e e d l e s (Monojet 202, 15 gauge, lh", b l u n t , on s t a n d a r d 3 c c s y r i n g e s ) . To p r e p a r e 102 ml o f c u l t u r e medium:  * 40 1 1 30 10 10 10  ml ml ml ml ml ml ml  2.2% m e t h y l c e l l u l o s e (Dow Chemicals) L-glutamine 29.2 mg/ml ( G e n e r a l B i o c h e m i c a l s 10510) 1 0 ~ M m e r c a p t o e t h a n o l (Baker Chemical Co. 08865) f e t a l c a l f serum (one l o t used throughout) b o v i n e serum albumin (Sigma A 4503)^ human l e u k o c y t e c o n d i t i o n e d medium e r y t h r o p o i e t i n a t 27.5 u n i t s / m l (epo, Step I I I , Connaught Laboratories) 2  * 2.2% m e t h y l c e l l u l o s e was made i n b a t c h e s o f 2 l i t r e s a c c o r d i n g t o the r o u t i n e p r o t o c o l used i n the l a b o r a t o r y o f . D r . Connie Eaves (70). **  P r e p a r e d a c c o r d i n g t o Gregory and E a v e s  Procedure:  (34).  J  1)  Mix 22 grams o f Dow s t a n d a r d grade m e t h y l c e l l u l o s e w i t h 22 grams o f Dow premium - grade m e t h y l c e l l u l o s e . Autoclave.  2)  Add the m i x t u r e o f m e t h y l c e l l u l o s e grades t o 1 l i t r e o f a u t o c l a v e d double d i s t i l l e d water a t 80°C i n a 2 - l i t r e E r l e n m e y e r f l a s k . Use  c o n t i n u o u s s t i r r i n g by magnetic  s t i r r e r w h i l e adding the powder.  3)  B o i l m i x t u r e f o r 1 minute  and l e t c o o l t o room  4)  Add  5)  S t i r a t 4°C o v e r n i g h t t o complete  6)  Dispense 40 ml a l i q u o t s and l e t s t a n d a t 20°C f o r 2 weeks as a s t e r i l i t y check.  7)  F r e e z e the a l i q u o t s f o r 24 h o u r s , then thaw i n the  8)  S t o r e a t 4°C.  1 l i t r e o f 2 x a-medium a t 37°C  (Connaught  temperature. Laboratories).  c l a r i f i c a t i o n process.  refrigerator.  

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