GastroenterologiaJaponica Vol. 14, No. 1 Copyright �9 1979 by The Japanese Society of Gastroenterology Printed in Japan

~ O r i g i n a l Art ic l e - -

RNase LEVELS IN G O L D E N HAMSTERS W I T H I N D U C E D PANCREATIC CANCER

D H P N

David W A L T O N , M.D. , Saburo N A K A Z A W A , M.D. , M a n a b u K A J I K A W A , M.D. ,

Yasuo N A I T O , M.D. , Masafumi I C H I K A W A , M.D. , Eizo K I M O T O , M.D. ,

Hiroshi SANO, M.D. , Yosuke SUZUKI, M.D. and

Toshio ASAI, M.D.

The Second Department of Internal Medicine, Nagoya University, School of Medicine

Tsuruma-cho, Showa-ku, Nagoya, 466Japan

Summary

Twenty Syrian golden hamsters received weekly injections of pancreatic cancer inducing DHPN. Their Poly (U) specific serum RNase levels were significantly elevated when compared to the control levels. Following salt fractionation, Poly (U) specific activity was present in both the 40% and 50% salt saturated fractions. Tissue assays showed that Poly (U) specific RNase was present in both pancreas and liver tissue extract, although the liver tissue RNase had a different pH maximum than that of the serum RNase.

Key Words: serum RNase, tissue RNase, experimental pancreatic cancer, DHPN, golden hamster.

Introduct ion

Because of the late appea rance of obvious

symptoms and the ambigu i ty involved in

present methods of differential diagnosis,

ca rc inoma of the pancreas is not only one of the

ma l ignan t tumors which is most difficult to

diagnose bu t its diagnosis is more of ten than

not too late. T h e low 5-year survival ra te for

pancrea t ic cancer pat ients (about 2 % ) led

Fraumeni t) to state that the "level of mor ta l i ty

is a good measure of incidence."

Reddi 2) demons t ra ted the corre la t ion

between pancrea t i c cancer and elevated serum

Received July 10, 1978. Accepted September 11, 1978. Address requests for reprints to: David Walton, M.D.,

2nd Department of Internal Medicine, Nagoya University School of Medicine, Tsuruma-Cho, Showa-ku, Nagoya, 466Japan.

The authors are grateful to Dr. Y. Hagino, Department of Pharmacology, Nagoya University School of Medicine for his valuable suggestions.

RNase levels in humans . D H P N (2,2'-

d ihydroxy-d i -N-propy ln i t rosamine) induced

pancrea t ic cancer in hamsters, which closely

resembles the ducta l adeno/zarcinoma found in

humans , has proven to be a useful model for

the morpholog ica l , histological, and secretory

studies of the pancreas undergo ing cancerous

growthS7). This report , a result of an in-

vestigation of serum and tissue RNase in

hamsters with pancrea t ic cancer , presents

evidence tha t elevated serum RNase levels

specific to secondary phospha te esters of

ur id ine 3 ' -phosphate were present in hamsters

with D H P N induced pancrea t ic cancer and

that an eleveted level was present in one

hamster after only 6 weeks of D H P N treat-

ment .

Materials and Methods

Reagents: D H P N was purchased from

February 1979 RNase Levels in DHPN Induced Pancreatic Cancer 75

Nakarai Chemicals, LTD. ; Poly (U) and Poly

(A) from Miles Labora tor ies , Inc.; Poly (G)

from Boehringer M a n n h e i m GmbH; Poly (C)

from Sigma Chemical Co. All other reagents

were of reagent g rade .

Subjects: 32 male Syrian golden hamsters,

with an average weight of 100 g, were used. 20

hamsters were given weekly injections of D H P N

( 5 0 0 m g / k g b.w.) unti l they were sacrificed;

The other 12 hamsters , who received no in-

jections, were used as controls. One t rea ted

animal was sacrificed at 6 weeks, three others at

14 weeks, and the rest f rom 20 to 30 weeks after

the start of t rea tment .

Serum Preparation: Blood, ob ta ined by

cut t ing the aor ta of anesthet ized animals, was

centr ifuged at 3000 RPM at room tempera tu re

for 20 minutes and the resul t ing superna tan t

was removed and saved.

Serum Assay: This p rocedure was essentially

the same as that descr ibed by Reddi s) with the

following minor a l terat ions:

1) React ion m i x t u r e -

0.10 ml of 6-fold d i lu ted serum

0 . 0 5 m l o f p o l y n u c l e o t i d e

(100 ug / .05 ml)

0 .10ml of phospha te -bora te buffer p H

7.0

2) React ion mixtures were incuba ted for 30

minutes.

3) Acid prec ip i ta ted mixtures were cen-

t r i fuged at 0 ~ for 30 minutes at 3000

RPM.

4) Superna tan ts were d i lu ted r0-fold and

absorbances measured at 260nm (Poly

(C) products were measured at 278 nm).

A m m o n i u m Sulfate Fractionation: After

0 .5ml of H20 was a d d e d to 0 .5ml of hamster

serum, a m m o n i u m sulfate was added to a 40%

satura t ion level, and the prec ip i ta te was

c o l l e c t e d by c e n t r i f u g a t i o n . S i m i l a r

precipi ta tes were collected for the 50%, 60%,

and 70% salt sa tura t ion levels. The 70%

sa tura ted superna tan t was also used. These

serum fractions were analyzed for their RNase

activity using the same procedure as that of the

s e r u m RNase assay ( f rac t ionated serum

samples were not d i lu ted) .

Tissue Extract: After resection of the

pancreas and the left lobe of the liver, an equal

weight of sea sand was a d d e d to each tissue and

this mixture was g round in a glass

homogenizer . Phospha te -bora te buffer, pH

6.5, was then a d d e d to this mix ture at a rat io o f

l ml bu f f e r / . 270g tissue. This mix ture was

homogenized and cent r i fuged at 12000xg for

30 minutes at 0 ~ The tissue assay procedure

was the same as the serum assay except that the

incuba t ion per iod was 15 minutes .

Protein Determinat ion: The color imetr ic

p rocedure of Lowry et al. s) was used.

Results

Pancrea t ic cancer was present in all animals

who received 20 weeks or more of D H P N

t r e a t m e n t ; d u c t a l h y p e r p l a s i a a n d

adenomatous a l terat ions were observed in all

animals receiving 14 weeks or more of DHPN

t rea tment (Fig. 1).

Serum RNase Assay: Hamster serum

exhib i ted the abil i ty to cleave secondary

phosphate esters of u r id ine 3 ' -phosphates and

Fig. I. Large adenocarcinoma of the pancreas; numerous papillary structures are present.

76 D. WALTON ET AL, Vol. 14, No. 1

cytidine 3'-phosphates. No activity was

exhibited towards Poly (A) and only very slight

activity towards Poly (G) (less than 5% that of

the Poly (U) or Poly (C) activity) was present.

The t ime course of hydrolysis (Fig. 2) shows the

pyrimidine base specificity of hamster serum

RNase. Hamster RNase exhibited m a x i m u m

activity at pH 7.0 towards both Poly (U) and

Poly (C) (Fig. 3).

Hamsters with pancreatic cancer exhibited

elevated ur idine base specific serum RNase

levels. The average (mean_S,E. ) of the ur id ine

specific RNase levels for the 20 DHPN treated

animals was 331+23 units while that for the

controls was 207+25 units. (A unit is defined as

that amount of RNase present in 0 .05ml of

diluted serum which caused a .001 rise in the

absorbance of acid solubilized assay products.)

The animals with the higher RNase levels were

not necessarily those that had been undergoing

t reatment the longest. There was no

correlation between the occurence of pan-

creatic cancer and abnormal cytidine specific

RNase levels (Fig. 4A and 4B).

A m m o n i u m S u l f a t e F r a c t i o n a t i o n : Uridine

specific activity was present in high levels in the

RNase Uni t . c 400

/ " / " Poly C

/ . , , ~ / / Poly(A) n ~ / / " adivity "

100

f /

TIME in Minutes

Fig. 2. Time course of hydrolysis. Aliquots were taken at 15, 30, 45 and 60 minutes. Poly (A) was unaffected by hamster serum RNase. The preference of hamster serum RNase for p y r i m i d i n e base homopolyribonucleotides is evident.

U n i t l

160

140

120

100

80

60

40

20

�9 ~ ~ P o l y U

poly c

I I I I

4.5 5.0 5 5 6.0 6.5 7.0 7.5 8.0 8.5 PH

Fig. 3. pH effect on the hydrolysis of Poly (U) and Poly (C) by hamster serum RNase. The buffers used were 0.1 M boric acid-sodium borate for pH values ranging from 4.5 to 5.5 and 0.1 M phosphate-borate buffer for values ranging from 6.0 to 8.5.

RNase Units X-260nm

450

300

150

Fig. 4A.

( P<'O. 001 )

g

O

-T- O �9

- i-

Controls DHPN Treated Hamsters (n-12) ( n-20 )

�9 6 wks wks

; 20 wks or more

Poly (U)-hamster serum RNase assay.

February 1979 R N a s e Levels in D H P N Induced Pancreat*b Cancer 77

40% and 50% salt precipitated serum frac-

tions. When comparing the treated animals' serum fractions to those of the controls, there was a general increase of RNase activity in the

two active fractions (40% and 50% salt precipitated); neither fraction increased

disproportionately to the other. For the Poly (C) fractionation assay all fractions exhibited

similar levels of RNase activity; no fractions

exhibited such a clear max imum activity as was present for the Poly (U) fractionation assay.

However, cytidine specific RNase levels were

greatly reduced when compared to the Poly (C)

activity prior to fractionation (Table 1). These results suggest that there is more than one pyrimidine specific enzyme. Perhaps also, the Poly (C) specific RNase operates in conjuction with an activator from which it was separated

o r an inhibitor which was activated during salt fractionation.

Inhibitors: The addition of 0.05ml of Poly

(A) or Poly (G) (100ug/ .05ml) to 0.05ml of Poly (U) caused a 90% and 97% reduction

respectively in the serum analysis for Poly (U)

active RNase (Table 2). The .use of boric acid-

borate buffer in place of the phosphate-borate

RNase Units X-T/Snm

800

600

400

Fig. 4B.

0

o

o

8 S

-I-- o o

8 8 ~

o �9 o !

I I - Controls DHPN Treated Hamsters ( n=12 ) ( n-20 )

~"~ 6wks

20 wks or more

Poly (C)-hamster serum RNase assay.

Table 1. Ammonium sulfate precipitated serum assay

Poly (U) assay DHPN

Control Treated

(n=5) (n=5)

Poly (C) assay DHPN

Control Treated

(n=5) (n=5)

units before fractionation 370 (-+43) 194 (-+20) 291 (-+65) 330 (• 8)

40%P 294 (-+36) 159 (• 32 (-+ 8) 27 (+ 6) 50%P 409 (• 214 (-+26) 39(-+ 5) 52(• 6) 60%P 47 (-+ 9) 43 (• 37 (-+ 6) 33 (• 8) 70%P 9(-+ 4) 4(-+ 2) 26(-+10) 28(• 70%S 72 (-+21) 47 (_+ 6) 35 (-+11) 25 (• 5)

%=per cent of salt saturation P=salt precipitated serum S=supernatant Values for the fractionated samples are the mean of the five ab- sorbances x 1000 -+ standard error.

78 D. WALTON ET AL. Vol. 14, No. 1

Table 2. Values given are the absorbance average • 1000

Inhibition by Poly(G)and Poly(A)

Only Poly (U) Poly (U) & (G) Poly (V) & (A)

Serum#1 519 40 10 Serum#2 505 47 13 Serum#3 307 37 10

Table 3. Pancreas and liver tissue extract assay

Assay

Tissue extract Pancreas Liver

DHPN DHPN Control Control

Treated Treated (n=2) (n=2)

(n=3) (n=3)

Poly (U) 714 766 302 393 Poly (C) 769 695 56 69 Poly (A) 269 283 78 94 Poly (G) 231 260 72 82

Values given are the absorbance average x 1000. The protein concentration of the pancreas and liver tissue extract were respectively 228 and 290 ug of protein per 0. t 0 ml of extract preparation.

buffer d id not al ter serum RNase Poly (U)

specific activity,

Tissue Ex t rac t Assay: Both pancreas and

liver tissue ext rac t exhib i ted an abi l i ty to cleave

all four homopolyr ibonucleot ides . Pancrea t ic

tissue ext rac t exhib i ted the highest RNase

levels for cleavage of Poly (U) and Poly (C).

Poly (U) cleavage by liver tissue ext rac t was less

than ha l f that of the pancrea t ic tissue, while

Poly (C) cleavage was less than 10% that of the

pancrea t ic tissue extract ( T a b l e 3).

Discussion

Like bovine serum, hamster serum possesses

RNase that is specific to the secondary

phosphate esters of ur id ine 3 ' -phospha te and

cytidine 3 ' -phosphate . Since these base specific

RNase activities are independen t of each other

(i.e. high Poly (U) RNase levels does not imply

that high Poly (C) RNase levels are also

present) and since their response after salt

f rac t ionat ion differs greatly, it appears that

these enzymes are distinct enzymes. Fur-

thermore , since Poly (U) at t ivi ty was present in

both the 40% and 50% salt p rec ip i t a ted

fractions, it is possible tha t there is more than

one Poly (U) specific enzyme.

Similar to the high levels of Poly (C) specific

RNase found in humans with pancrea t ic

cancer, e levated levels of Poly (U) specific

RNase were present in hamsters with pan-

creat ic cancer . Also s imilar to the presence of

Poly (C) specific enzyme found in h u m a n

pancrea t ic tissueS), Poly (U) specific RNase was

present in hamster pancrea t ic tissue extract .

Poly (U) specific enzyme was also present in

liver tissue extract , however it had an acidic pH

m a x i m u m (pH 4 .5-5 .5) and its RNase level was

less than ha l f the value of the pancrea t i c tissue

extract .

February 1979 RNase Levels in D H P N Induced Pancreatic Cancer 79

I t is i m p o r t a n t to n o t e t h a t n e o p l a s t i c g r o w t h

was p r e s e n t in t he l ivers o f all t r e a t e d a n i m a l s

e x c e p t for t h e 6 weeks t r e a t e d h a m s t e r . D H P N ,

w h i c h ha s in t he pas t i n d u c e d c a r c i n o m a o f t h e

k idneys in h a m s t e r s , p r o d u c e d n o such les ions

in t h e h a m s t e r s u sed fo r th i s s tudy .

O f spec ia l i n t e r e s t is t h e f ac t t h a t a n e l e v a t e d

Poly (U) specif ic R N a s e level was p r e s e n t in o n e

h a m s t e r a f t e r on ly six weeks o f D H P N t r e a t -

m e n t . Due to t he c o r r e s p o n d e n c e b e t w e e n t h e

o c c u r r e n c e o f p a n c r e a t i c c a n c e r a n d e l e v a t e d

s e r u m levels in h u m a n s a n d h a m s t e r s , p e r h a p s

e l e v a t e d s e r u m R N a s e levels c o u l d serve as a

b i o c h e m i c a l i n d i c a t o r o f ea r ly p a n c r e a t i c

c a n c e r .

References

1) Fraumeni JFJr: Cancers of the pancreas and biliary

tract: epidemotogical considerations. Cancer Res 35: 3437-3446, 1975

2) Reddi KK, Holland JF: Elevated serum ribonuclease in patients with pancreatic cancer. Proc Natl Acad Sci USA 73: 2308-2310, 1976

3) Nakazawa S, et al: Experimental study of the pan- creatic carcinoma induced by the administration of di-isnpropanolnitrosamine. Jpn J Gastroenterol 74: 581 588, 1977

4) Kajikawa M: Studies of DHPN induced pancreatic carcinoma with emphasis of pancreatography. Jpn J Gastroenterol 75:1386 1398, 1978

5) Pour P, et al: Cancer of the pancreas induced in the Syrian golden hamster. Am J Pathol 76: 349-358, 1974

6) Pour P, et al: A new approach for induction of pancreatic neoplasms. Cancer Res 35: 2259-2268, 1975

7) Reber HA, et al: Pancreatic secretion in hamsters with pancreatic cancer. Surgery 82: 34-41, 1977

8) Reddi KK: Nature and 'possible origin of human serum ribonuclease. Biochem Biophys Res Commun 67: 110-118, 1975