- Email: [email protected]
In vitro effects of methotrexate on human osteoblasts
688
Abstracts
Bone Vol. 16, No. 6 June 1 9 9 5 : 6 7 9 - 6 9 5
Mean:~em (n 12)
Normal Proliferating
Normal Hypertrophic
TD Proliferatin8
Kd (pM) Receptors/cell
29.8+3.3 1344-+186
39.5:t:3.1 1706-+201
33.6+4.9 1439-+120
TD Lesion 83.4+95.6 913_+92
N o r m a l c h o n d r o c y t e s f r o m the h y p e r t r o p h i c z o n e h a v e a h i g h e r VDR n u m b e r t h a n t h o s e f r o m the p r o l i f e r a t i n g z o n e (p<0.01, p a i r e d t-test), a l t h o u g h the affinity of 1.25 for VDR is similar. C h o n d r o c y t e s f r o m the p r o l i f e r a t i n g z o n e of TD affected birds s h o w e d n o s i g n i f i c a n t d i f f e r e n c e in e i t h e r VDR n u m b e r or affinity compared to n o r m a l b i r d s . H o w e v e r , lesion chondrocytes h a v e b o t h a r e d u c e d VDR n u m b e r a n d affinity (p<0.01, D u n n ' s test) c o m p a r e d to all o t h e r zones. Since 1,25 is k n o w n to induce its o w n receptor these results may offer one e x p l a n a t i o n of w h y t r e a t m e n t of birds with 1,25 can prevent TD. Funded by a link grant from the BBSRC
P30. M i l k f o r t i f i e d w i t h v i t a m i n D3 and calcium: safety a n d efficacy in young adults MJ M c K e n n a , R F r e a n e y , P Byrne, Y McBrinn, B M u r r a y , M Kelly, B Donne, M O'Brien
St. Vincent's Hospital, St.Michael's Hospital, Department of Physiology, University College Dublin, Department of Public Dental Health and Community Services, Dublin Dental ftospital, Department of Anatomy, Dublin University, Dublin We s t u d i e d the safety a n d efficacy of fortified milk in y o u n g a d u l t s ( m e d i a n a g e 22.6 yr; range: 18-48 yr). Fortification increased the vitamin D3 content from 0.3 , g / L to 12 , g / L , a n d elemental c a l c i u m f r o m 1270 m g / L to 1525 m g / L . Volunteers (123) w e r e recruited; 102 c o m p l e t e d the trial. A c c o r d i n g to a d o u b l e - b l i n d p r o t o c o l they w e r e assigned in a r a n d o m m a n n e r to receive e i t h e r fortified m i l k or u n f o r t i f i e d milk. Milk c o n s u m p t i o n w a s not different b e t w e e n groups: 65+40 litres in fortified g r o u p vs 64+_21 litres in non- fortified group. BlocKt was d r a w n in late a u t u m n a n d after the winter. Paired samples were m e a s u r e d in the s a m e a s s a y . S e a s o n a l d e c r e a s e in serunl 25(OH)D w a s s i g n i f i c a n t l y d i m i n i s h e d in the g r o u p d r i n k i n g fortified milk. In the fortified g r o u p , s e r u m 25(OH)D decreased b y 15 n m o l / L f r o m 77+35 to 62_+26 n m o l / L (P<0.001). In the control g r o u p , s e r u m 25(OH)D fell by 31 n m o l / L from 85-+39 to 54-+25 n m o l / L (P<0.00I). Both g r o u p s h a d a slight but significant rise in s e r u m ionised calcium, corrected for pH: fortified g r o u p (1.24_+0.04 vs 1.27-+0.06 m m o l / L ; P<.001); control g r o u p (1.23_+0.03 vs 1.26+0.03 m m o l / L ; P<.O01)). We p r o p o s e that fortification of milk with vitamin D be m a n d a t o r y in EU states.
P31. Bone loss after the menopause: cross-sectlonal studies m a y be m i s l e a d i n g PA Mole, CR Paterson
Department of Biochemical Medicine, Ninewells Hospital and Medical School, Dundee DDI 9SY We r e p o r t o n a s e v e n y e a r s t u d y of b o n e d e n s i t y in 67 p o s t m e n o p a u s a l w o m e n . U s i n g the s a m e M o l s g a a r d single p h o t o n a b s o r p t i o m e t e r ND1100A t h r o u g h o u t , w e m e a s u r e d the f o r e a r m b o n e m i n e r a l content (BMC). Four to ten (median 6) BMC m e a s u r e m e n t s over p e r i o d s of five to seven (median 6 9 ) years were obtained. The rate of b o n e loss calculated from the s t u d y p o p u l a t i o n as a cross-section w a s high in y o u n g e r w o m e n , one to nine years past the m e n o p a u s e , b u t m u c h lower in w o m e n more than ten years p o s t m e n o p a u s a l . H o w e v e r the m e a n l o n g i t u d i n a l rates of loss in i n d i v i d u a l s w e r e f o u n d to be no different in the t w o a g e groups. The l o n g i t u d i n a l rates of c h a n g e did not c h a n g e with a g e or m e n o p a u s a l age. A w o m a n t w e n t y years after the m e n o p a u s e could be losing b o n e at the s a m e rate as one five years after the menopause. We s u g g e s t that care should be taken in m a k i n g inferences for the future from cross-sectional data. W o m e n n o w in their sixties m i g h t a p p e a r from cross- sectional data to be losing bone more slowly t h a n is actually the case, as s h o w n by the longitudinal d a t a . H o r m o n e r e p l a c e m e n t t h e r a p y m a y well be w o r t h c o n s i d e r i n g at a n y age not just in the first few years after the menopause.
P32. Broadband ultrasound characteristics in rheumatoid arthritis and diabetes D De Lord, S P u t h r a s i n g a m , P Mulligan, K Brown, G Waiters, P Pitt
Farnborough Hospital, Orpington, Kent Introduction: B r o a d b a n d u l t r a s o u n d is currently being evaluated as a simple, n o n - i o n i s i n g investigation of bone in patients with s u s p e c t e d o s t e o p o r o s i s . R e d u c e d b r o a d b a n d ultrasorgnd at a p e r i p h e r a l site m a y be a m e a s u r e of altered b o n e structure, r e f l e c t i n g s y s t e m i c b o n e loss a n d p r e d i c t i n g f r a c t u r e risk. M e t h o d s : M e a s u r e m e n t s of b r o a d b a n d u l t r a s o u n a t t e n u a t i o n (BUA), speed of s o t m d (SOS) a n d 'stiffness', a derivative of these values, w e r e t a k e n at the os calcis in 50 w o m e n with RA, 58 d i a b e t i c s a n d a n a g e - m a t c h e d n o r m a l p o p u l a t i o n . Possible influencing factors w e r e assessed by questionnaire. Results: BUA, SOS a n d stiffness w e r e significantly lower in RA g r o u p t h a n controls: stiffness a g e - m a t c h e d z score correlated significantly w i t h H A Q score (disability index), r=-0.41, p
Institutes of Pathology, Erasmus University, Rotterdam, The Netherlands and *Aarhus University, Denmark, **Department of Internal Medicine III, University Hospital Dijkzigt, 3015 GD Rotterdam, The Netherlands W h e n b o n e r e m o d e l l i n g is m a r k e d l y elevated, lack of completed w a l l s p r e c l u d e s e s t i m a t i o n of s e v e r a l p a r a m e t e r s of b o n e t u r n o v e r , s u c h as d u r a t i o n of the f o r m a t i v e period (FP) a n d a c t i v a t i o n f r e q u e n c y (Ac.f), as o n e e s s e n t i a l p a r a m e t e r , c o m p l e t e d w a l l w i d t h (W.Wi) c a n n o t be assessed. Recently, Steiniche et al, p u b l i s h e d a m e t h o d to select a n d m e a s u r e walls not yet c o m p l e t e l y m i n e r a l i s e d b u t c o m p l e t e d as to their thickness (Bone 1992, 13: 147): these are f o u n d in the lowest quartile of the v a l u e s of the ratio osteoid w i d t h / m i n e r a l i z e d width. W e u s e d this criterion, p r i m a r i l y developed to s t u d y the effect of intervention, to reconstruct W.Wi (rW.Wi) in a d v a n c e d renal o s t e o d y s t r o p h y (ROD). W.Wi w a s r e c o n s t r u c t e d in c a n c e l l o u s b o n e f r o m lilac crest biopsy sections from 12 patients with predialysis ROD a n d 12 agea n d s e x - m a t c h e d controls; null hypothesis was tested b y MannW h i t n e y ' s U-test: ROD rW.Wi FP ll.Mlt Ac.f
~m days days /year
43.1 119.6 32.8 1.7
controls averages 58.9 86.6 12.5 0.9
p <0.03 < 0.03 < 0.01 < 0.005
ROD, apart from an impaired T h u s , in p r e d i a l y s i s m i n e r a l i s a t i o n ( i n c r e a s e d initial m i n e r a l i s a t i o n lag t i m e ll.Mlt), we f o u n d walls to be s u b n o r m a l l y thin. While activation f r e q u e n c y w a s f o u n d increased, a p r o t r a c t e d formative period had to be inferred.
P34. In vitro effects of methotrexate on h u m a n osteoblasts BAA Scheven*, MJ v a n d e r Veen, CA D a m e n , FPJG Lafeber, HJM v a n Rijn, JWJ Bijlsma, SA D u u r s m a
University Hospital Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands and *Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB Methotrexate l o w - d o s e t h e r a p y is considered to be a favourable a p p r o a c h for the t r e a t m e n t of r h e u m a t o i d arthritis (RA). To
Bone Vol. 16, No. 6 June 1 9 9 5 : 6 7 9 - 6 9 5
i n v e s t i g a t e w h e t h e r MTX has d i r e c t effects o n o s t e o b l a s t s , p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n of h u m a n t r a b e c u l a r b o n e d e r i v e d osteoblast-like cells w e r e assessed after culture in the p r e s e n c e of v a r i o u s d o s e s of MTX in 5% F B S - m e d i u m either s u p p l e m e n t e d or n o t w i t h 10 nM 1 , 2 5 - d i h y d r o x y v i t a m i n D3 (1,25(0H)2D3). T r e a t m e n t of the osteoblastic cells w i t h MTX resulted in a h i g h l y significant, d o s e - d e p e n d e n t inhibition of cell p r o l i f e r a t i o n w i t h half m a x i m a l r e s p o n s e at a d o s e of 30 nM. C o m p l e t e b l o c k a g e of D N A synthesis at MTX doses of >100 nM c o r r e s p o n d e d w i t h a n a p p r o x i m a t e 30% r e d u c t i o n in cell n u m b e r a f t e r 4 d a y s ' of culture. MTX d i d not alter cellular a l k a l i n e p h o s p h a t a s e (AP) a c t i v i t y , the n u m b e r of cells e x p r e s s i n g c y t o c h e m i c a l AP, or b a s a l osteocalcin p r o d u c t i o n . A d d i t i o n of 1,25(OH)2D3 to the cultures caused an e n h a n c e d AP e x p r e s s i o n (~ t w o f o l d ) a n d osteocalcin p r o d u c t i o n (~ 30-fold) c o i n c i d i n g w i t h a d e c r e a s e d o s t e o b l a s t p r o l i f e r a t i o n . Coi n c u b a t i o n of 1,25(OH)2D3 with MTX in doses > 100 nM further decreased cell n u m b e r s b y a b o u t 10%, a n d i n d u c e d a significant s t i m u l a t i o n (30-40%) of A P activity as well as p r o d u c t i o n of osteocalcin (= 20%) a b o v e the values reached in the 1,25(OH)2D3 cultures. In c o n c l u s i o n . MTX at low R A - t h e r a p e u t i c a l levels p r o v e d to be a p o t e n t i n h i b i t o r of o s t e o b l a s t proliferation, h o w e v e r , MTX d i d not affect b a s a l osteoblastic p h e n o t y p i c e x p r e s s i o n . In the p r e s e n c e of the o s t e o b l a s t differentiationp r o m o t e r . 1,25(OH)2D3, MTX further inhibited cell proliferation w h i c h w a s associated w i t h e n h a n c e d AP activity a n d osteocalcin p r o d u c t i o n . T h u s , MTX m a y h a v e p r o f o u n d effects o n b o n e r e m o d e l l i n g a n d m e t a b o l i s m b y i n t e r f e r i n g w i t h b o n e cell turnover.
P35. I n s u l i n - l i k e growth factor (IGF-I) is not a parathyroid hormone-related protein secretagogue in goat milk IR Fleet, SK Abbas*, A D Care*
AFRC Institute of Animal Physiology and Genetic Research, Babraham, Cambridge CB2 4AT and *Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DD P a r a t h y r o i d h o r m o n e - r e l a t e d p r o t e i n (PTHrP), a p u t a t i v e c a l c i u m m o b i l i s i n g f a c t o r of l a c t a t i o n is f o u n d in h i g h c o n c e n t r a t i o n s in m i l k f r o m a v a r i e t y of m a m m a l s . In a n a t t e m p t to i d e n t i f y its s e c r e t a g o g u e s in m i l k , 6 g o a t s in e s t a b l i s h e d lactation w e r e i n f u s e d for 6 h via a m a m m a r y arterial catheter w i t h IGF-I or saline on different days. Milk a n d m a m m a r y v e n o u s b l o o d w a s collected at 1-2 h interval for 8 h d u r i n g the c o n t r o l (2 h) a n d infusion periods. Milk f r o m b o t h sides of the g l a n d w a s collected w i t h the aid of i.v oxytocin (100 m U / m l ) a n d m i l k y i e l d r e c o r d e d . The c o n c e n t r a t i o n s of P T H r P ( | - 3 4 ) in m i l k a n d p l a s m a w e r e m e a s u r e d b y a r a d i o i m m u n o a s s a y w h i c h e m p l o y e d a rabbit PTHrP(1-34) a n t i s e r u m , PTHrP(1-34) as s t a n d a r d a n d i o d i n a t e d - [ T y r ° ] - I r F H r P ( 1 - 3 4 ) as tracer. Milking the c o n t r o l g o a t s at h o u r l y intervals over 8 h w i t h the aid of oxytocin led to an increase in the Pl'HrP(1-34) c o n c e n t r a t i o n in the milk w i t h o u t d e p r e s s i o n of the milk yield. H o w e v e r , close i n t r a m a m m a r y arterial infusion of IGF-I (150 n m o l / h ) for 6 h into g o a t s c a u s e d no significant increase in VTHrP (1o34) concentration in the m i l k (7.9 + 4.5 n m o l / l ; m e a n _+ S.E.M) relative to t h a t associated with the infusion of saline (2.4 + 0.8 n m o l / l ) over the s a m e period. Plasma P T H r P (1-34) also r e m a i n e d u n c h a n g e d in b o t h g r o u p s over the 8 h period. We c o n c l u d e that IGP-I d o e s not a p p e a r to be a significant P T H r P s e c r e t a g o g u e in the milk of goats.
P36. Apoptosis in growing rat bone: preliminary studies BS Noble, J Milne*, N Loveridge*"
Bone Research Group, Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen AB2 2ZD, *Biochemical Sciences, Rowett Institute, Aberdeen AB2 9SB and **Bone Research Group (MRC), Department of Medicine, Addenbrooke's Hospital, Cambridge CB2 2QQ A p o p t o s i s , is c h a r a c t e r i s e d b y n u c l e a r c o n d e n s a t i o n , D N A f r a g m e n t a t i o n , cell m e m b r a n e b l e b b i n g a n d r a p i d r e m o v a l w i t h o u t a n i n f l a m m a t o r y response. Recent reports indicate a correlation b e t w e e n D N A f r a g m e n t a t i o n in situ a n d apoptosis. O t h e r characteristics i n c l u d i n g c - m y c a n d c-fos expression a n d i n c r e a s e s in D N A s y n t h e s i s w i t h o u t s u b s e q u e n t cell
Abstracts
p r o l i f e r a t i o n h a v e also b e e n associated with apoptosis. Since b o t h o n c o g e n e e x p r e s s i o n a n d D N A s y n t h e s i s w i t h o u t cell proliferation h a v e b e e n reported in various b o n e cell types a n d osteoblast a n d c h o n d r o c y t e cell d e a t h h a v e been associated with m i n e r a l i s a t i o n w e have investigated the possible presence of DNA f r a g m e n t a t i o n in sections of b o n e from y o u n g (4 w e e k old) rats. D N A s t r a n d b r e a k s w e r e d e m o n s t r a t e d b y D I G - d U T P labelling the 3' h y d r o x y l termini with terminal transferase (TdT) or D N A p o l y m e r a s e I. I n c o r p o r a t e d DIG-dUTP w a s detected b y FITC or p e r o x i d a s e c o n j u g a t e d second antibodies. Preliminary e v i d e n c e s u g g e s t s t h a t D N A s t r a n d b r e a k s o c c u r in cortical osteocytes, a few cells on e n d o s t e a l , periosteal a n d trabecular b o n e surfaces a n d within the b o n e m a r r o w a n d in g r o w t h plate c h o n d r o c y t e s . D N A s e t r e a t m e n t resulted in m o s t nuclei being labelled w h i l e o m i s s i o n of TdT or p o l y m e r a s e resulted in the absence of staining. There were n o differences between the t w o m e t h o d s . These s t u d i e s s u g g e s t t h a t a p o p t o s i s m a y occur in s o m e b o n e cells a n d as s u c h m a y h a v e i m p o r t a n t implications for o u r u n d e r s t a n d i n g of the c o n t r o l of b o n e f o r m a t i o n a n d resorption.
P37. H u m a n breast cancer cell lines express the PTHIPTHrP receptor MA Birch, JA C a r r o n , W D Fraser*, JA Gallagher
Departments of Human Anatomy and Cell Biology and *Clinical Chemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX P a r a t h y r o i d h o r m o n e - r e l a t e d protein (PTHrP) is p r o d u c e d b y a v a r i e t y of cell t y p e s a n d s h a r e s m a n y biological effects with p a r a t h y r o i d h o r m o n e ( P T H ) b y b i n d i n g to a c o m m o n [ Y r H / P T H r P receptor. P T H r P has been s h o w n to be p r o d u c e d b y breast c a n c e r cells b o t h in vioo a n d in vitro a n d is k n o w n to be an i m p o r t a n t m e d i a t o r of h y p e r c a l c a e m i a of malignancy. In this s t u d y w e h a v e c o n s i d e r e d the possible role of IYI'HrP in the aetiology of breast cancer, a n d h a v e examined the expression of the 17rH/IYI'HrP receptor in a panel of h u m a n breast cancer cell lines. U s i n g RT-PCR followed b y S o u t h e r n blot a n a l y s i s w e detected [YI'H/[Y]'HrP receptor m R N A in MCF-7, T-47-D, SKBr-3, Hs578T a n d MDA-MB231 cells. R N A from MCF-7 cells w a s analysed b y N o r t h e r n h y b r i d i s a t i o n with a 17]'H/IYI'HrP receptor p r o b e a n d s h o w e d specific t r a n s c r i p t s of 1.5kb a n d 2.4kb. In proliferation studies, MCF-7 cells i n c o r p o r a t e d 3 H - t h y m i d i n e in r e s p o n s e to e x o g e n o u s P T H a n d P T H r P (1-34) in a d o s e d e p e n d e n t m a n n e r (1.6-100ng/ml), i n d i c a t i n g that the PTH receptor is functional in these cells. Hs578T cells, w h i c h did not express the r e c e p t o r , d i d n o t r e s p o n d to e x o g e n o u s IYI'H or [Y]'HrP. MCF-7 cells also s h o w e d increased levels of intracellular cyclic A M P in r e s p o n s e to 17YHrP. The expression of functional IYI'H receptor b y breast cancer cell lines suggests that [rFHrP m a y be i n v o l v e d in a u t o c r i n e o r p a r a c r i n e r e g u l a t i o n of b r e a s t t u m o u r g r o w t h a n d m e t a s t a s i s in vivo.
P38. T h r o m b o s p o n d i n p r o m o t e s resorption by osteoclasts JA C a r r o n , CA Walsh, W D Fraser*, JA Gallagher
isolated
Departments of Human Anatomy and Cell Biology and *Clinical Chemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX We h a v e investigated the effects of both t h r o m b o s p o n d i n (TSP) a n d fibronectin (Fn) on resorption of devitalised slices of s p e r m w h a l e d e n t i n e b y isolated a v i a n osteoclasts. Dentine slices were soaked in a solution of Fn or TSP (both 2 0 u g / m l ) or in buffer o v e r n i g h t , w a s h e d a n d seeded with a mixed p o p u l a t i o n of bone cells. A f t e r 3hrs settling, the slices w e r e rinsed briefly a n d resorption a l l o w e d to proceed in m e d i u m at 37°C for 24hrs. At the e n d of the r e s o r p t i o n period, the slices w e r e examined for total a d h e r e n t cell n u m b e r s a n d for total plan area of resorption. While Pn-soaked d e n t i n e h a d a large (4-5 fold) increase in both a t t a c h e d cell n u m b e r s a n d area of r e s o r p t i o n o v e r control dentine, TSP-soaked slices s h o w e d twice the level of resorption of c o n t r o l s w i t h o u t a n y a p p a r e n t increase in a t t a c h e d cell n u m b e r s . Dentine slices s o a k e d in a mixture of TSP a n d Fn h a d twice as m u c h resorption as those soaked in Fn alone, a n d eight times the control levels. Since slices soaked in Fn alone w e r e
689
Abstracts
Bone Vol. 16, No. 6 June 1 9 9 5 : 6 7 9 - 6 9 5
Mean:~em (n 12)
Normal Proliferating
Normal Hypertrophic
TD Proliferatin8
Kd (pM) Receptors/cell
29.8+3.3 1344-+186
39.5:t:3.1 1706-+201
33.6+4.9 1439-+120
TD Lesion 83.4+95.6 913_+92
N o r m a l c h o n d r o c y t e s f r o m the h y p e r t r o p h i c z o n e h a v e a h i g h e r VDR n u m b e r t h a n t h o s e f r o m the p r o l i f e r a t i n g z o n e (p<0.01, p a i r e d t-test), a l t h o u g h the affinity of 1.25 for VDR is similar. C h o n d r o c y t e s f r o m the p r o l i f e r a t i n g z o n e of TD affected birds s h o w e d n o s i g n i f i c a n t d i f f e r e n c e in e i t h e r VDR n u m b e r or affinity compared to n o r m a l b i r d s . H o w e v e r , lesion chondrocytes h a v e b o t h a r e d u c e d VDR n u m b e r a n d affinity (p<0.01, D u n n ' s test) c o m p a r e d to all o t h e r zones. Since 1,25 is k n o w n to induce its o w n receptor these results may offer one e x p l a n a t i o n of w h y t r e a t m e n t of birds with 1,25 can prevent TD. Funded by a link grant from the BBSRC
P30. M i l k f o r t i f i e d w i t h v i t a m i n D3 and calcium: safety a n d efficacy in young adults MJ M c K e n n a , R F r e a n e y , P Byrne, Y McBrinn, B M u r r a y , M Kelly, B Donne, M O'Brien
St. Vincent's Hospital, St.Michael's Hospital, Department of Physiology, University College Dublin, Department of Public Dental Health and Community Services, Dublin Dental ftospital, Department of Anatomy, Dublin University, Dublin We s t u d i e d the safety a n d efficacy of fortified milk in y o u n g a d u l t s ( m e d i a n a g e 22.6 yr; range: 18-48 yr). Fortification increased the vitamin D3 content from 0.3 , g / L to 12 , g / L , a n d elemental c a l c i u m f r o m 1270 m g / L to 1525 m g / L . Volunteers (123) w e r e recruited; 102 c o m p l e t e d the trial. A c c o r d i n g to a d o u b l e - b l i n d p r o t o c o l they w e r e assigned in a r a n d o m m a n n e r to receive e i t h e r fortified m i l k or u n f o r t i f i e d milk. Milk c o n s u m p t i o n w a s not different b e t w e e n groups: 65+40 litres in fortified g r o u p vs 64+_21 litres in non- fortified group. BlocKt was d r a w n in late a u t u m n a n d after the winter. Paired samples were m e a s u r e d in the s a m e a s s a y . S e a s o n a l d e c r e a s e in serunl 25(OH)D w a s s i g n i f i c a n t l y d i m i n i s h e d in the g r o u p d r i n k i n g fortified milk. In the fortified g r o u p , s e r u m 25(OH)D decreased b y 15 n m o l / L f r o m 77+35 to 62_+26 n m o l / L (P<0.001). In the control g r o u p , s e r u m 25(OH)D fell by 31 n m o l / L from 85-+39 to 54-+25 n m o l / L (P<0.00I). Both g r o u p s h a d a slight but significant rise in s e r u m ionised calcium, corrected for pH: fortified g r o u p (1.24_+0.04 vs 1.27-+0.06 m m o l / L ; P<.001); control g r o u p (1.23_+0.03 vs 1.26+0.03 m m o l / L ; P<.O01)). We p r o p o s e that fortification of milk with vitamin D be m a n d a t o r y in EU states.
P31. Bone loss after the menopause: cross-sectlonal studies m a y be m i s l e a d i n g PA Mole, CR Paterson
Department of Biochemical Medicine, Ninewells Hospital and Medical School, Dundee DDI 9SY We r e p o r t o n a s e v e n y e a r s t u d y of b o n e d e n s i t y in 67 p o s t m e n o p a u s a l w o m e n . U s i n g the s a m e M o l s g a a r d single p h o t o n a b s o r p t i o m e t e r ND1100A t h r o u g h o u t , w e m e a s u r e d the f o r e a r m b o n e m i n e r a l content (BMC). Four to ten (median 6) BMC m e a s u r e m e n t s over p e r i o d s of five to seven (median 6 9 ) years were obtained. The rate of b o n e loss calculated from the s t u d y p o p u l a t i o n as a cross-section w a s high in y o u n g e r w o m e n , one to nine years past the m e n o p a u s e , b u t m u c h lower in w o m e n more than ten years p o s t m e n o p a u s a l . H o w e v e r the m e a n l o n g i t u d i n a l rates of loss in i n d i v i d u a l s w e r e f o u n d to be no different in the t w o a g e groups. The l o n g i t u d i n a l rates of c h a n g e did not c h a n g e with a g e or m e n o p a u s a l age. A w o m a n t w e n t y years after the m e n o p a u s e could be losing b o n e at the s a m e rate as one five years after the menopause. We s u g g e s t that care should be taken in m a k i n g inferences for the future from cross-sectional data. W o m e n n o w in their sixties m i g h t a p p e a r from cross- sectional data to be losing bone more slowly t h a n is actually the case, as s h o w n by the longitudinal d a t a . H o r m o n e r e p l a c e m e n t t h e r a p y m a y well be w o r t h c o n s i d e r i n g at a n y age not just in the first few years after the menopause.
P32. Broadband ultrasound characteristics in rheumatoid arthritis and diabetes D De Lord, S P u t h r a s i n g a m , P Mulligan, K Brown, G Waiters, P Pitt
Farnborough Hospital, Orpington, Kent Introduction: B r o a d b a n d u l t r a s o u n d is currently being evaluated as a simple, n o n - i o n i s i n g investigation of bone in patients with s u s p e c t e d o s t e o p o r o s i s . R e d u c e d b r o a d b a n d ultrasorgnd at a p e r i p h e r a l site m a y be a m e a s u r e of altered b o n e structure, r e f l e c t i n g s y s t e m i c b o n e loss a n d p r e d i c t i n g f r a c t u r e risk. M e t h o d s : M e a s u r e m e n t s of b r o a d b a n d u l t r a s o u n a t t e n u a t i o n (BUA), speed of s o t m d (SOS) a n d 'stiffness', a derivative of these values, w e r e t a k e n at the os calcis in 50 w o m e n with RA, 58 d i a b e t i c s a n d a n a g e - m a t c h e d n o r m a l p o p u l a t i o n . Possible influencing factors w e r e assessed by questionnaire. Results: BUA, SOS a n d stiffness w e r e significantly lower in RA g r o u p t h a n controls: stiffness a g e - m a t c h e d z score correlated significantly w i t h H A Q score (disability index), r=-0.41, p
Institutes of Pathology, Erasmus University, Rotterdam, The Netherlands and *Aarhus University, Denmark, **Department of Internal Medicine III, University Hospital Dijkzigt, 3015 GD Rotterdam, The Netherlands W h e n b o n e r e m o d e l l i n g is m a r k e d l y elevated, lack of completed w a l l s p r e c l u d e s e s t i m a t i o n of s e v e r a l p a r a m e t e r s of b o n e t u r n o v e r , s u c h as d u r a t i o n of the f o r m a t i v e period (FP) a n d a c t i v a t i o n f r e q u e n c y (Ac.f), as o n e e s s e n t i a l p a r a m e t e r , c o m p l e t e d w a l l w i d t h (W.Wi) c a n n o t be assessed. Recently, Steiniche et al, p u b l i s h e d a m e t h o d to select a n d m e a s u r e walls not yet c o m p l e t e l y m i n e r a l i s e d b u t c o m p l e t e d as to their thickness (Bone 1992, 13: 147): these are f o u n d in the lowest quartile of the v a l u e s of the ratio osteoid w i d t h / m i n e r a l i z e d width. W e u s e d this criterion, p r i m a r i l y developed to s t u d y the effect of intervention, to reconstruct W.Wi (rW.Wi) in a d v a n c e d renal o s t e o d y s t r o p h y (ROD). W.Wi w a s r e c o n s t r u c t e d in c a n c e l l o u s b o n e f r o m lilac crest biopsy sections from 12 patients with predialysis ROD a n d 12 agea n d s e x - m a t c h e d controls; null hypothesis was tested b y MannW h i t n e y ' s U-test: ROD rW.Wi FP ll.Mlt Ac.f
~m days days /year
43.1 119.6 32.8 1.7
controls averages 58.9 86.6 12.5 0.9
p <0.03 < 0.03 < 0.01 < 0.005
ROD, apart from an impaired T h u s , in p r e d i a l y s i s m i n e r a l i s a t i o n ( i n c r e a s e d initial m i n e r a l i s a t i o n lag t i m e ll.Mlt), we f o u n d walls to be s u b n o r m a l l y thin. While activation f r e q u e n c y w a s f o u n d increased, a p r o t r a c t e d formative period had to be inferred.
P34. In vitro effects of methotrexate on h u m a n osteoblasts BAA Scheven*, MJ v a n d e r Veen, CA D a m e n , FPJG Lafeber, HJM v a n Rijn, JWJ Bijlsma, SA D u u r s m a
University Hospital Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands and *Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB Methotrexate l o w - d o s e t h e r a p y is considered to be a favourable a p p r o a c h for the t r e a t m e n t of r h e u m a t o i d arthritis (RA). To
Bone Vol. 16, No. 6 June 1 9 9 5 : 6 7 9 - 6 9 5
i n v e s t i g a t e w h e t h e r MTX has d i r e c t effects o n o s t e o b l a s t s , p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n of h u m a n t r a b e c u l a r b o n e d e r i v e d osteoblast-like cells w e r e assessed after culture in the p r e s e n c e of v a r i o u s d o s e s of MTX in 5% F B S - m e d i u m either s u p p l e m e n t e d or n o t w i t h 10 nM 1 , 2 5 - d i h y d r o x y v i t a m i n D3 (1,25(0H)2D3). T r e a t m e n t of the osteoblastic cells w i t h MTX resulted in a h i g h l y significant, d o s e - d e p e n d e n t inhibition of cell p r o l i f e r a t i o n w i t h half m a x i m a l r e s p o n s e at a d o s e of 30 nM. C o m p l e t e b l o c k a g e of D N A synthesis at MTX doses of >100 nM c o r r e s p o n d e d w i t h a n a p p r o x i m a t e 30% r e d u c t i o n in cell n u m b e r a f t e r 4 d a y s ' of culture. MTX d i d not alter cellular a l k a l i n e p h o s p h a t a s e (AP) a c t i v i t y , the n u m b e r of cells e x p r e s s i n g c y t o c h e m i c a l AP, or b a s a l osteocalcin p r o d u c t i o n . A d d i t i o n of 1,25(OH)2D3 to the cultures caused an e n h a n c e d AP e x p r e s s i o n (~ t w o f o l d ) a n d osteocalcin p r o d u c t i o n (~ 30-fold) c o i n c i d i n g w i t h a d e c r e a s e d o s t e o b l a s t p r o l i f e r a t i o n . Coi n c u b a t i o n of 1,25(OH)2D3 with MTX in doses > 100 nM further decreased cell n u m b e r s b y a b o u t 10%, a n d i n d u c e d a significant s t i m u l a t i o n (30-40%) of A P activity as well as p r o d u c t i o n of osteocalcin (= 20%) a b o v e the values reached in the 1,25(OH)2D3 cultures. In c o n c l u s i o n . MTX at low R A - t h e r a p e u t i c a l levels p r o v e d to be a p o t e n t i n h i b i t o r of o s t e o b l a s t proliferation, h o w e v e r , MTX d i d not affect b a s a l osteoblastic p h e n o t y p i c e x p r e s s i o n . In the p r e s e n c e of the o s t e o b l a s t differentiationp r o m o t e r . 1,25(OH)2D3, MTX further inhibited cell proliferation w h i c h w a s associated w i t h e n h a n c e d AP activity a n d osteocalcin p r o d u c t i o n . T h u s , MTX m a y h a v e p r o f o u n d effects o n b o n e r e m o d e l l i n g a n d m e t a b o l i s m b y i n t e r f e r i n g w i t h b o n e cell turnover.
P35. I n s u l i n - l i k e growth factor (IGF-I) is not a parathyroid hormone-related protein secretagogue in goat milk IR Fleet, SK Abbas*, A D Care*
AFRC Institute of Animal Physiology and Genetic Research, Babraham, Cambridge CB2 4AT and *Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DD P a r a t h y r o i d h o r m o n e - r e l a t e d p r o t e i n (PTHrP), a p u t a t i v e c a l c i u m m o b i l i s i n g f a c t o r of l a c t a t i o n is f o u n d in h i g h c o n c e n t r a t i o n s in m i l k f r o m a v a r i e t y of m a m m a l s . In a n a t t e m p t to i d e n t i f y its s e c r e t a g o g u e s in m i l k , 6 g o a t s in e s t a b l i s h e d lactation w e r e i n f u s e d for 6 h via a m a m m a r y arterial catheter w i t h IGF-I or saline on different days. Milk a n d m a m m a r y v e n o u s b l o o d w a s collected at 1-2 h interval for 8 h d u r i n g the c o n t r o l (2 h) a n d infusion periods. Milk f r o m b o t h sides of the g l a n d w a s collected w i t h the aid of i.v oxytocin (100 m U / m l ) a n d m i l k y i e l d r e c o r d e d . The c o n c e n t r a t i o n s of P T H r P ( | - 3 4 ) in m i l k a n d p l a s m a w e r e m e a s u r e d b y a r a d i o i m m u n o a s s a y w h i c h e m p l o y e d a rabbit PTHrP(1-34) a n t i s e r u m , PTHrP(1-34) as s t a n d a r d a n d i o d i n a t e d - [ T y r ° ] - I r F H r P ( 1 - 3 4 ) as tracer. Milking the c o n t r o l g o a t s at h o u r l y intervals over 8 h w i t h the aid of oxytocin led to an increase in the Pl'HrP(1-34) c o n c e n t r a t i o n in the milk w i t h o u t d e p r e s s i o n of the milk yield. H o w e v e r , close i n t r a m a m m a r y arterial infusion of IGF-I (150 n m o l / h ) for 6 h into g o a t s c a u s e d no significant increase in VTHrP (1o34) concentration in the m i l k (7.9 + 4.5 n m o l / l ; m e a n _+ S.E.M) relative to t h a t associated with the infusion of saline (2.4 + 0.8 n m o l / l ) over the s a m e period. Plasma P T H r P (1-34) also r e m a i n e d u n c h a n g e d in b o t h g r o u p s over the 8 h period. We c o n c l u d e that IGP-I d o e s not a p p e a r to be a significant P T H r P s e c r e t a g o g u e in the milk of goats.
P36. Apoptosis in growing rat bone: preliminary studies BS Noble, J Milne*, N Loveridge*"
Bone Research Group, Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen AB2 2ZD, *Biochemical Sciences, Rowett Institute, Aberdeen AB2 9SB and **Bone Research Group (MRC), Department of Medicine, Addenbrooke's Hospital, Cambridge CB2 2QQ A p o p t o s i s , is c h a r a c t e r i s e d b y n u c l e a r c o n d e n s a t i o n , D N A f r a g m e n t a t i o n , cell m e m b r a n e b l e b b i n g a n d r a p i d r e m o v a l w i t h o u t a n i n f l a m m a t o r y response. Recent reports indicate a correlation b e t w e e n D N A f r a g m e n t a t i o n in situ a n d apoptosis. O t h e r characteristics i n c l u d i n g c - m y c a n d c-fos expression a n d i n c r e a s e s in D N A s y n t h e s i s w i t h o u t s u b s e q u e n t cell
Abstracts
p r o l i f e r a t i o n h a v e also b e e n associated with apoptosis. Since b o t h o n c o g e n e e x p r e s s i o n a n d D N A s y n t h e s i s w i t h o u t cell proliferation h a v e b e e n reported in various b o n e cell types a n d osteoblast a n d c h o n d r o c y t e cell d e a t h h a v e been associated with m i n e r a l i s a t i o n w e have investigated the possible presence of DNA f r a g m e n t a t i o n in sections of b o n e from y o u n g (4 w e e k old) rats. D N A s t r a n d b r e a k s w e r e d e m o n s t r a t e d b y D I G - d U T P labelling the 3' h y d r o x y l termini with terminal transferase (TdT) or D N A p o l y m e r a s e I. I n c o r p o r a t e d DIG-dUTP w a s detected b y FITC or p e r o x i d a s e c o n j u g a t e d second antibodies. Preliminary e v i d e n c e s u g g e s t s t h a t D N A s t r a n d b r e a k s o c c u r in cortical osteocytes, a few cells on e n d o s t e a l , periosteal a n d trabecular b o n e surfaces a n d within the b o n e m a r r o w a n d in g r o w t h plate c h o n d r o c y t e s . D N A s e t r e a t m e n t resulted in m o s t nuclei being labelled w h i l e o m i s s i o n of TdT or p o l y m e r a s e resulted in the absence of staining. There were n o differences between the t w o m e t h o d s . These s t u d i e s s u g g e s t t h a t a p o p t o s i s m a y occur in s o m e b o n e cells a n d as s u c h m a y h a v e i m p o r t a n t implications for o u r u n d e r s t a n d i n g of the c o n t r o l of b o n e f o r m a t i o n a n d resorption.
P37. H u m a n breast cancer cell lines express the PTHIPTHrP receptor MA Birch, JA C a r r o n , W D Fraser*, JA Gallagher
Departments of Human Anatomy and Cell Biology and *Clinical Chemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX P a r a t h y r o i d h o r m o n e - r e l a t e d protein (PTHrP) is p r o d u c e d b y a v a r i e t y of cell t y p e s a n d s h a r e s m a n y biological effects with p a r a t h y r o i d h o r m o n e ( P T H ) b y b i n d i n g to a c o m m o n [ Y r H / P T H r P receptor. P T H r P has been s h o w n to be p r o d u c e d b y breast c a n c e r cells b o t h in vioo a n d in vitro a n d is k n o w n to be an i m p o r t a n t m e d i a t o r of h y p e r c a l c a e m i a of malignancy. In this s t u d y w e h a v e c o n s i d e r e d the possible role of IYI'HrP in the aetiology of breast cancer, a n d h a v e examined the expression of the 17rH/IYI'HrP receptor in a panel of h u m a n breast cancer cell lines. U s i n g RT-PCR followed b y S o u t h e r n blot a n a l y s i s w e detected [YI'H/[Y]'HrP receptor m R N A in MCF-7, T-47-D, SKBr-3, Hs578T a n d MDA-MB231 cells. R N A from MCF-7 cells w a s analysed b y N o r t h e r n h y b r i d i s a t i o n with a 17]'H/IYI'HrP receptor p r o b e a n d s h o w e d specific t r a n s c r i p t s of 1.5kb a n d 2.4kb. In proliferation studies, MCF-7 cells i n c o r p o r a t e d 3 H - t h y m i d i n e in r e s p o n s e to e x o g e n o u s P T H a n d P T H r P (1-34) in a d o s e d e p e n d e n t m a n n e r (1.6-100ng/ml), i n d i c a t i n g that the PTH receptor is functional in these cells. Hs578T cells, w h i c h did not express the r e c e p t o r , d i d n o t r e s p o n d to e x o g e n o u s IYI'H or [Y]'HrP. MCF-7 cells also s h o w e d increased levels of intracellular cyclic A M P in r e s p o n s e to 17YHrP. The expression of functional IYI'H receptor b y breast cancer cell lines suggests that [rFHrP m a y be i n v o l v e d in a u t o c r i n e o r p a r a c r i n e r e g u l a t i o n of b r e a s t t u m o u r g r o w t h a n d m e t a s t a s i s in vivo.
P38. T h r o m b o s p o n d i n p r o m o t e s resorption by osteoclasts JA C a r r o n , CA Walsh, W D Fraser*, JA Gallagher
isolated
Departments of Human Anatomy and Cell Biology and *Clinical Chemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX We h a v e investigated the effects of both t h r o m b o s p o n d i n (TSP) a n d fibronectin (Fn) on resorption of devitalised slices of s p e r m w h a l e d e n t i n e b y isolated a v i a n osteoclasts. Dentine slices were soaked in a solution of Fn or TSP (both 2 0 u g / m l ) or in buffer o v e r n i g h t , w a s h e d a n d seeded with a mixed p o p u l a t i o n of bone cells. A f t e r 3hrs settling, the slices w e r e rinsed briefly a n d resorption a l l o w e d to proceed in m e d i u m at 37°C for 24hrs. At the e n d of the r e s o r p t i o n period, the slices w e r e examined for total a d h e r e n t cell n u m b e r s a n d for total plan area of resorption. While Pn-soaked d e n t i n e h a d a large (4-5 fold) increase in both a t t a c h e d cell n u m b e r s a n d area of r e s o r p t i o n o v e r control dentine, TSP-soaked slices s h o w e d twice the level of resorption of c o n t r o l s w i t h o u t a n y a p p a r e n t increase in a t t a c h e d cell n u m b e r s . Dentine slices s o a k e d in a mixture of TSP a n d Fn h a d twice as m u c h resorption as those soaked in Fn alone, a n d eight times the control levels. Since slices soaked in Fn alone w e r e
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