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Differentiation — dependent binding of EGF to human colon adenocarcinomata
352
HUNAN PREPROGLUCAGONCOHPONENT PEPTIDES ARE TOPOGRAPHICALLY SEGREGATED W[THIN THE PANCREATIC ALPHA GRANULE
I.M. V a r n d e l l l , K.L. S i k r i l , S.R. Bloom2 and J . t l . Polakl Depts. of i H i s t o c h e m i s t r y and 2Medicine, Royal Postgraduate Medical School, Hammersmith H o s p i t a l , LONDON WI20HS, U.K. The s t r u c t u r e of the human preproglucagon molecule has r e c e n t l y been deduced from n u c l e o t i d e sequencing of cDNA derived from p a n c r e a t i c tumour RNA. Two h i t h e r t o unknown p e p t i d e s , g l u c a g o n - l i k e peptides (GLPI, GLP2), were found to form a C-terminal extension t o , but separated from, g l i c e n t i n - a 69 amino acid molecule which contains glucagon w i t h i n i t s s t r u c t u r e . Previous r e p o r t s have described " t r u e " glucagon ( i . e . having a free C-terminus) which has a s t r i k i n g sequence s i m i l a r i t y to GLPI , to be r e s t r i c t e d t o the e c c e n t r i c e l e c t r o n - d e n s e core of the p a n c r e a t i c alpha g r a n u l e , whereas g l i c e n t i n was l o c a l i s e d e x c l u s i v e l y to the h a l o . Using an antiserum d i r e c t e d t o GLPI ( r a b b i t p o l y c l o n a l , d i l u t i o n 1:16,000) which showed no c r o s s - r e a c t i v i t y w i t h glucagon (up t o 20 nmol peptide/m] d i l u t e d antiserum) in the immuno- cytochemical system employed we have been able t o demonstrate GLPI s o l e l y in the e l e c t r o n - d e n s e core, using c o l l o i d a l gold probe immunoelectron microscopy. Immunoreactive s i t e s were v i s u a l i s e d using goat a n t i - r a b b i t IgG adsorbed onto i 0 or 20 nm gold p a r t i c l e s . In accord w i t h previous f i n d i n g s g l i c e n t i n appears to be r e s t r i c t e d to the h a l o . An antibody raised t o glucagon16,29, p r e v i o u s l y believed t o be s p e c i f i c f o r " t r u e " glucagon, was found t o c r o s s - r e a c t e x t e n s i v e l y w i t h GLPI and t o be t o t a l l y adsorbed by t h i s p e p t i d e in low ( 0 . 5 - 1 . 0 nmol/ml) concentration. In c o n c l u s i o n , i t would seem t h a t GLPI in a d d i t i o n t o , or even r a t h e r than, p a n c r e a t i c glucagon, is r e s t r i c t e d t o the core of the alpha g r a n u l e . This i s a s i g n i f i c a n t f i n d i n g in the understanding of glucagon N o s y n t h e s i s and s e c r e t i o n .
DIFFERENTIATION DEPENDENT ADENOCARCINOMATA
BINDING
OF
EGF
TO
HUMAN
COLON
ALLISON F WREN and 3.B. ELDER, Department of Surgery, Manchester University Medical School, Manchester, UK.
The role of EQF in controlling the growth of mucosal tissues is not well documented. However, observations that EGF can inhibit gastric acid secretion and increase the rate of healing of experimentally-induced gastric and duodenal ulcers has confirmed the target organ status of the G.I. tract. We have used crude membrane fractions from normal human colon mucosa (n = 5) and from solid tumours of this region (n =18) to investigate the EGF binding of these tissues. The fractions were prepared by differential centrifugation and aliquots containing 100 ~Jg protein incubated with varying concentrations of 125I-EGF. From Scatchard analysis of the binding data, EGF binding to normal mucosa varies from least in the ascending colon (0.03 pM/mg protein) to most in the sigmoid (0.29 pM/mg protein). Tumours in all areas bound less EGF than normal mucosa. However, the a f f i n i t y o~ ~,e cancerous tissue was higher; K for normal des~nding colonic mucosa was 4.5 x 10 - M whereas that for tumour tissue was 8.0 x tO- M. Tumour histology was examined and the adenocarcinomas divided into well and poorly differentiated types. Further analysis of the binding data showed that the welldifferentiated turnouts bound significantly more EGF than poorly differentiated cancers originating in the colon area. We conclude that it may be possible to use EGF binding as a tumour marker for some types of neoplasia.
HUNAN PREPROGLUCAGONCOHPONENT PEPTIDES ARE TOPOGRAPHICALLY SEGREGATED W[THIN THE PANCREATIC ALPHA GRANULE
I.M. V a r n d e l l l , K.L. S i k r i l , S.R. Bloom2 and J . t l . Polakl Depts. of i H i s t o c h e m i s t r y and 2Medicine, Royal Postgraduate Medical School, Hammersmith H o s p i t a l , LONDON WI20HS, U.K. The s t r u c t u r e of the human preproglucagon molecule has r e c e n t l y been deduced from n u c l e o t i d e sequencing of cDNA derived from p a n c r e a t i c tumour RNA. Two h i t h e r t o unknown p e p t i d e s , g l u c a g o n - l i k e peptides (GLPI, GLP2), were found to form a C-terminal extension t o , but separated from, g l i c e n t i n - a 69 amino acid molecule which contains glucagon w i t h i n i t s s t r u c t u r e . Previous r e p o r t s have described " t r u e " glucagon ( i . e . having a free C-terminus) which has a s t r i k i n g sequence s i m i l a r i t y to GLPI , to be r e s t r i c t e d t o the e c c e n t r i c e l e c t r o n - d e n s e core of the p a n c r e a t i c alpha g r a n u l e , whereas g l i c e n t i n was l o c a l i s e d e x c l u s i v e l y to the h a l o . Using an antiserum d i r e c t e d t o GLPI ( r a b b i t p o l y c l o n a l , d i l u t i o n 1:16,000) which showed no c r o s s - r e a c t i v i t y w i t h glucagon (up t o 20 nmol peptide/m] d i l u t e d antiserum) in the immuno- cytochemical system employed we have been able t o demonstrate GLPI s o l e l y in the e l e c t r o n - d e n s e core, using c o l l o i d a l gold probe immunoelectron microscopy. Immunoreactive s i t e s were v i s u a l i s e d using goat a n t i - r a b b i t IgG adsorbed onto i 0 or 20 nm gold p a r t i c l e s . In accord w i t h previous f i n d i n g s g l i c e n t i n appears to be r e s t r i c t e d to the h a l o . An antibody raised t o glucagon16,29, p r e v i o u s l y believed t o be s p e c i f i c f o r " t r u e " glucagon, was found t o c r o s s - r e a c t e x t e n s i v e l y w i t h GLPI and t o be t o t a l l y adsorbed by t h i s p e p t i d e in low ( 0 . 5 - 1 . 0 nmol/ml) concentration. In c o n c l u s i o n , i t would seem t h a t GLPI in a d d i t i o n t o , or even r a t h e r than, p a n c r e a t i c glucagon, is r e s t r i c t e d t o the core of the alpha g r a n u l e . This i s a s i g n i f i c a n t f i n d i n g in the understanding of glucagon N o s y n t h e s i s and s e c r e t i o n .
DIFFERENTIATION DEPENDENT ADENOCARCINOMATA
BINDING
OF
EGF
TO
HUMAN
COLON
ALLISON F WREN and 3.B. ELDER, Department of Surgery, Manchester University Medical School, Manchester, UK.
The role of EQF in controlling the growth of mucosal tissues is not well documented. However, observations that EGF can inhibit gastric acid secretion and increase the rate of healing of experimentally-induced gastric and duodenal ulcers has confirmed the target organ status of the G.I. tract. We have used crude membrane fractions from normal human colon mucosa (n = 5) and from solid tumours of this region (n =18) to investigate the EGF binding of these tissues. The fractions were prepared by differential centrifugation and aliquots containing 100 ~Jg protein incubated with varying concentrations of 125I-EGF. From Scatchard analysis of the binding data, EGF binding to normal mucosa varies from least in the ascending colon (0.03 pM/mg protein) to most in the sigmoid (0.29 pM/mg protein). Tumours in all areas bound less EGF than normal mucosa. However, the a f f i n i t y o~ ~,e cancerous tissue was higher; K for normal des~nding colonic mucosa was 4.5 x 10 - M whereas that for tumour tissue was 8.0 x tO- M. Tumour histology was examined and the adenocarcinomas divided into well and poorly differentiated types. Further analysis of the binding data showed that the welldifferentiated turnouts bound significantly more EGF than poorly differentiated cancers originating in the colon area. We conclude that it may be possible to use EGF binding as a tumour marker for some types of neoplasia.