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Electrophysiological studies on the cultured cells obtained from transplantable pancreatic carcinoma in Syrian golden hamsters
Pepnde,~. Vol 5. pp 423---428.1984. ' Ankho InternationalInc Pnnted m the U.S.A.
0196-9781/84$3 00 + O0
Electrophysiological Studies on the Cultured Cells Obtained From Transplantable Pancreatic Carcinoma in Syrian Golden Hamsters N. UEDA, Y. SUZUKI, M. UTSUMI, T. OBARA, K. OKAMURA AND M. NAMIKI Third D e p a r t m e n t o f Internal Medicine, A s a h i k a w a M e d i c a l College 3-11, 4-5, NishiAagura, A s a h i k a w a , J a p a n
UEDA, N., Y. SUZUKI, M. UTSUMI. T. OBARA, K. OKAMURA AND M. NAMIK1. Electrophysiological .~tudw.~on the t ultured cells obtained frmn transplantable pancreatw carcinoma in Syrian golden hamster~. PEPTIDES 5(2) 423-428, 1984.--A pancreatic ductal carcinoma was established as a transplantable tumor line in an inbred strain of Syrian golden hamsters. Intracellular recordings of membrane potentials and input resistance were made from cultured cells obtained from the transplanted tumors using indwelling glass microelectrode. The mean value of the resting membrane potential was -46.5_+ 1.8 mV (S.E.) In= 13), while the mean resting input resistance was 21.2±4.3 MI) (S.E.) (N=I3). Dibutyryl cyclic AMP f2 x 10-a M) caused a marked hyperpolarization of about 30 mV accompanied by a reduction of input resistance. The transplantable tumor and Its cultured cell line developed in this study have demonstrated their effectiveness as a reliable experimental model for use m pancreatic cancer research. Pancreatic ductal carcinoma Syrian golden hamster Transplantable pancreatic cancer Effect of secretin. VIP. and dibutyryl cyclic AMP Membrane potential Input resistance N-nltrosobts [2-hydroxypropyl] amine
P A N C R E A T I C cancer in humans has been on the increase during this century, taking an annual toll of about 8,000 in Japan. Moreover. because the disease is usually in its advanced stages upon discovery, there is a very low five-year survival rate [5]. A pancreatic ductal adenocarcinoma induced by N-nitrosobis (2-hydroxypropyl) amine (DHPN) in the Syrian golden hamster is remarkably similar to that occurring in man m its morphoioglc characteristics [4,12]. Therefore, the Syrian hamster is a useful model to study pancreatic cancer. Recently we succeeded in establishing a transplantable tumor line in an inbred strain of Syrian hamsters which appears to be suitable for use in further biological and therapeutic investigations. The present investigation was concerned with the bioelectrical properties of the cultured cells obtained from the tumor transplanted m the Syrian golden hamsters.
Following a period of progressive weight loss (between 30-40 weeks after initial treatment), the hamsters were killed by cervical dislocation. Pancreatic tumors were removed under sterile conditions and divided into two parts, one for transplantation and the other for histological examination.
Transplantation The tumor tissue was placed in a sterile petri dish with Hanks balanced salt solution. The tumor was minced into small pieces with scissors (about I mm cubes), and one or two fragments of the tumor tissue were inoculated subcutaneously with a trocar into the interscapular area of 4 to 6-week-old male syngeneic hamsters. Serial transplantations were performed in the same manner. Recipient hamsters were killed 6 weeks after the transplantation, and a part of the tumor tissue was transplanted serially.
Cell culture
METHOD
For the electrophysiological studies, a cell culture was made with a transplant from the twenty-fifth passage. The tumor was removed from a cutaneous incision, minced into fine fragments and incubated with RPMI 1640 medium containing 10% fetal calf serum and 0.1% collagenase (from Sigma) for l hr at 37°C with shaking.
TIIIllOI" hldllcfioll Eight-week-old male Syrian golden hamsters (Nisseiken Co.. Tokyo. Japan) were injected subcutaneously with DHPN in 0.9% NaCI solution at a dose of 250 mg/kg body wetght once weekly for 20 weeks.
423
424
UEDA ET AL.
FIG. I. Histoio~ fmdim~ of tm'hna_rytumor showins well dil~erendaled ~ t k
Individual cells and clump cells were passed through a free mesh, while coarse fibrous tissues and debris were remined in the mesh. The cells were washed three times with RPMI 1640 medium. The cell suspension (4× 10-s ceils/ml) was cultured for 1 week, in 95~ air and 5% CO, at 37~C. Measurement of Membrane Potential and Resistance
The culture plate (5 ml) was perfused with the standard Krebs-H_em~_it solution warmed to 37eC at a constant rate of 2.5 ml/min. The staadard solution had the following composition (raM): NaLl 103, KCI 4.7, CaLl 2.56, M41CI 1.13, NaHCOs 25, NaHPO( 1.15, D-glucose 2.8, Na pyruvate 4.9, Na 81uatmate 4.9, Na fitmarate 2.7. It was Iptased with 95% 02 5% CO~. Glass micmeiectro~s (1.5 mm in outer diameter) were pulled by a m k z o e l e c u ~ puller ( N ~ , Japan) and [i!kd with 3 M-KCI. The electrode had a tip resistance of about 15-20 MQ. Measurements of membrane potential and resistance were carried out by one intracelhflar microelectrode which was connected to an electrometer amplifier (W-P KS 700) allowins current ~ n and meamrement of membrane potential and resistance. Stimulants were added directly to the tissue bath close to the impaled microelectrode using a fine needle connected to a syrinlle. Sec~in and VII) were stored at -20*C and dissolved in a buffer solution just before application.
ductai cell carcinoma. (H and Ex 120.)
The drugs used were: VIP (Protein Research Foundation, Japan), secretin (Eisai, Japan) synthesized caendein (Kyowa Hakko, Japan), acetyicholine chloride (Wako chemicals, Japan), dibutyryl cyclic AMP (Sigma), cyclic AMP (Sigma). RESULTS Tumors were induced in all of the hamsters administered repeated injections of DHPN. Hiatoiogkal examinatioas of the original tumor showed typical features of welldifferentiated l x m c ~ duct adenocaeciaoma (Fig. I). The tran~lented tumors became psipsble at the inoculated site after 1 week and thereafter iprew ~ y . The tumors were suceessf~y passed through 30 Ileneratiorm, and the tnmspimttability rate was 67~ to 100~. The doublial; time of the tumor volume varied from 3.4 to 5.7 days in the 1lth to 18th generations, and the average doubling time was 4.1_.0.82 days. Histolollieal fmdialls of the transplaated tumor (25th generation) revealed it to be similar to those of the otis'hal tumor (Fig. 2). Culture cells of the transplanted tumor exhibited typical characteristics of an epithelial with larse nuclei, and the 1 or 2 small nu¢leoli were observed (Fig. 3). The mean resting membrane potential obtained from stable impalements comtinued for more than 3 mim,'tes was -46.$__.1.8 mV (S.E.) (n=13). The current-voltalle relationship was obtained by injecting rectanselar c.rref~s through
425
FIG. 2. Micro~cpic section from the twenty-fi/~h generation demonstrating the same h/stolosical appearance as the orig/nal tumor. (H and Exl20.)
FIG. 3. One week-old cultured cells. The cells are cuboidal and grow in sheets which are tightly adimrent to each other. (Phase contrast, original magnification x400.)
U E D A ET AL
426 VtP
MP(mv)
a KCI b
:
I I
i
1
mm
-~ 0 I -I° I -30
-2
-!
!025hA
-40
•
i
-$0
I 0 25 nA
i xlO-=A
-60 ,-';'0 50 ms
FIG. 5. The effect of VIP and KCI on the membrane potential and resistance. The top part shows a pen recording. 'vii) (0.35 V,g) (to obtain an estimated peak concentration of at least 2x 10-' M} did not cause any effect on the membrane potential and resistance, while an application of KCI (3.2 rag) (to achieve an estimated peak concentration of about 20 raM) caused an immediate depolarization. The lower part shows the time course ofelectrotonic potential change before and during the action of KCI.
-80 -90
I TOmV dbc~AP
I
a
m¢
~-] o b
¢ --
ii
i|
, , .... .
-4OmV
50ms
FIG. 4. The resting current-voltnge relattonship m pancreattc cancer cells. The lower parts shows membrane potential (taP) changes in response to recta,,tgular I~0 ms¢c current pulses of differcm magnitude. The top part shows plot of membrane potential as a function of magnitude and polarity of injected current.
]
I0
the recording electrode. The relation was almost linear between - 1 7 and - 8 6 mV (Fig. 4), which corresponds to an input resistance o f about 20 MI"L The mean resting input resistance within the range o f the linear current-voltage relation was 21.2_+4.3 Mfl (S.E.) ( n = 13). An application o f V1P (0.35 ~g) did not cause any c h a n p o f membrane potential and resistance, wh/le in tim same cell a direct addition o f KCI (3.2 rag) in order to augment K ion concentration resulted in a marked depolarization o f 45 mV accompanied by a slight reduction of input resistance (Fig. 5). In subscglutmt experiments, secretin (0.15 #g), acetylcholine (0.9 t~g), and caerulein (0.8 ng) had no effects on the membrane potential and resistance. Figure 6 shows the effect o f dibutyryl cyclic A M P (4 rag). Application o f dibutyryl cyclic A M P cammd a marked hyperlmlarization from - 3 3 mV to - 6 7 mV accompanied by the reduction of input resistance and shortening of time constant. The same results were obtained in 3 experiments. The mean membrane potential at the peak o f hyperpolarization induced by dibutyryl cyclic A M P was - 6 8 . 0 _+2.9 mV (S.E.) (n=3). tn contrast to the marked hyperpolarizing effect o f dibutyryl cyclic AMP, cyclic AMP, which is less lipophilic, caused a smaller hyperpolarization o f only 5 mV followed by a marked depolarization of 15 inV. DISCUSSION
The failure o f est',d~lishing on appropriate experimental ammal model for the study o f pancreatic cancer has resulted
, b
i
L
"
0 5nA
gnA
v ' -
F
e~ocal
FIG. 6. The effect of dibutyryl cyclic AMP (dbc AMP) on the membrane potential and resistance. The top part shows a pen recording Addition of dbc AMP (4 rag) to the culture plate to achieve an estimated peak concentration of at least 2×10 -~ M dunng intracelValar recordings caused m a r k e d hyperlmlarization accompanied by the reduction of input resistance. The lower part shows the time course of elcctrotonic p o t e n t i a l change before (a). during (b) and after (c) the action of dbc AMP.
in considerable delays in this field as compared to studtes on other gastrointestinal tract cancers. In 1974 Pour (,t (d. succeeded in inducing pancreatic duct adenocarcinoma in Syrian golden hamsters by chronic systemic adminhtlration of DHPN. Since this induced pancreatic cancer is quite similar morphologically to human ductal adenocarcinoma, their model appears to be suttable for use in the study o f pancreattc cancer {7, I I].
TRANSPLANTABLE PANCREATIC CANCER
427
Recently several reports on the establishment of transplantable tumor lines in inbred strains of Syrian hamsters have been published [6, 15, 17, 20, 21]. We also have succeeded in establishing a transplantable tumor line in our laboratory. The transplantable tumor demonstrated comparably fast growth and small possibility o f metastasis as well as histological similarity to human well-differentiated ductal adenocarcinoma. In addition, we confirmed that the histological findings of transplantable tumor are almost identical to those found in primary pancreatic cancer. These evidence support the usefulness o f this tumor in chemotherapeutic studies of pancreatic cancer as well as in biological and biochemical investigations. While morphological studies o f experimental pancreatic cancer have advanced, biological studies especially from the electrophysioiogical approach have not yet been conducted. In order to investigate the bioelectrical properties of pancreatic cancer cells, we have established a cell culture of transplantable tumor, and using these cells, have performed measurements o f membrane potentials and input reststance changes under stimuli of VIP, secretin, caerulein, acetylcholine, and dibutyryl cyclic AMP. The mean resting cell membrane potential was - 4 6 . 5 inV. This value is quite similar to already reported resting membrane potentials of several exocrine gland cells ( - 4 0 ~ - 7 0 mV) [9,19]. The current-voltage relationship was almost linear and exhibited a slight outward going rectification. This result indicated that the membrane conductance o f this cell is dominated by K + and C1-. As shown in Fig. 4, the increase o f K ÷ concentration in the perfusate caused a marked depolarization of the membrane potential, which further confirmed the essential role of K + ion in membrane conductance. Secretin and VIP, which are known as physiological stimulants o f the pancreatic ductal cell, had no effect on the membrane potenttai and input resistance in the cultured cancer cells in this study. Furthermore, potent stimulants of the pancreatic acinar cell such as acetylcholine and caerulein
also had no effect on them. However, admimstration of a penetrating analogue of cyclic A M P , which is thought to be an intraceilular mediator o f the effect of secretin and VIP on HCO..7 secretion in the pancreatic ductal cells, evoked a marked hyperpolarization accompanied by a reduction of input resistance [I, 2, 10, 16, 18]. Binding studies using J-Or'l-secretin in pancreatic cancer cells of hamsters and humans suggested the presence of secretin receptors on the membrane of these cells [3,15]. However, in our electrophysiological study, secretin or VIP had no apparent effect on the membrane potential or input resistance of the tumor cells, which indicates impaired membrane function incident to the neoplastic transformation. There is relatively little information about the effect of cyclic A M P on electrical properties of cell membranes. It has been demonstrated that glucagon evoked membrane hyperpolarization in liver parenchymal cells was mimicked by cyclic AMP, which is known as an intracellular mediator of the effect ofglucagon [8,14]. However, there is no available evidence on the effect of cyclic A M P on the electrical properties of tumor cell membranes. The effect of cyclic A M P will not, however, be finally solved until the effect o f secretin or VIP as well as cyclic A M P has been tested on an electrical properties o f those cells where the physiologically important receptors are probably localized (the centro-acinar or terminal duct cells). In conclusion, the transplantable tumor and its cultured cell line developed in this study have demonstrated their effectiveness as a reliable experimental model for use in pancreatic cancer research.
ACKNOWLEDGEMENT This work was supported by a Grant-in-Aid for Special Project Research from the Ministry of Education, Science and Culture. We thank Miss J. Watanabe for typing the manuscript and Miss C. Asai for technical assistance.
REFERENCES
I. Case. R. M. and T Scratcherd. The actions ofdibutyryl cyclic adenosine 3'.5'-monophosphate and methylxanthines on pancreatic exocrine secretion. J Phvsiol 223: 649--667, 1972. 2 Deschodt-Lanckman, M., P. Robberecht. P. De Neef and J. Christophe. In vitro action of bombesm and bombesin-like pepudes on amylase secretion, calcmm effiux, and adenylate cyclase acuvlty in the rat pancreas. J Clin lnve,~t 58: 891-898, 1976. 3. Estival, A.. F, Clemente and A. Ribet. Adenocarcinoma of the human exocrine pancreas: Presence of secretin and caerulein receptors, Biothem Biophvs Re~ Commun 102: 1336--1341, 1981. 4 Flaks, A.. M. A. Moor and B. Flaks. Ultrastructural analysis of pancreatic carcinogenesis. 11 Establishment and morphology of a transplantable hamster pancreatic adenocarcinoma. FCP. Cart im~genesis l: 513-522. 1980. 5. Hnrayama. T. Epidemiological study on pancreatic cancer. (m Japanese). Kan Tan sui 4: 477---487. 1982. 6 Komshi. Y . S. TakahashL M. Sunagawa. N. Mnyagt and H. Kondo. Heterotransplantation into nude mice of pancreatnc carcinoma reduced by N-bis (2-hdyroxypropyl) nitrosamine in hamsters Can~ er Lett 5: 333--337, 1978.
7. Levitt, M. H., C. C, Harris, R. Squire, S. Spnnger, M. Wenk, C. Mollelo, D. Thomas, E. Kingsbury and C. Newkirk. Experimental pancreatic carcinogensis. I. Morphogenesls of pancreatnc adenoearcinoma in the syrian golden hamster induced by N-nitroso-bis (2-hydroxypropyl) amine. Am J Pathol 88: 5-15, 1977. 8. Petersen, O. H. The effect of glucagon on the liver cell membrane potential. J Physiol/-~9: 647--656, 1974. 9. Petersen, O. H. The Electrophysiology o f Gland Cells. New York: Academic Press, 1980. 10. Petersen, O. H. and N. U~la. Secretion of fluid and amylase in the perfused rat pancreas. J Physiol 2,64: 819--835, 1977. 11. Pour, P., F. W. K~ger and J. Althoff. Cancer of the pancreas induced in the Syrian golden hamster. Am J Pathol 76: 349-358. 1974. 12. Pour, P., J. Althoff and M. Takahashi. Early lesions of pancreatic ductal carcinoma in the hamster model. A m J Pathol 88: 291-303, 1977. 13. Pour, P., R. R. Runge, D. Birt, R. Gingell, T. Lawson. D. Nagel, L. Wallcave and S. Z. Salmasn. Pancreatic carcmogensis in the hamster and its relevance to the human disease. Cant er 47: 1573-1587, 1981.
428 14. Robison, G. A.. R. W. Butcher and E. W. Sutherland. Cyclic AMP. New York: Academic Press, 1971. 15. ScarpeUi, D. G. and M. S. Rao. Transplantable ductal adenocarcinoma of the Syrian hamster pancreas. Cancer Res 39: 452-458, 1979, 16. Schulz, I., K. Heil. A. Kdbben, G. Sachs and W. Haase. Isolation and functional characterization of cells from the exocrine pancreas. In: Biology o f Normal and Cancerous Exocrine Pancreatic Cells, edited by A. Ribet, L. Praoayrol and C. Sugini. New York: Elsevier/North-Holland Biochemical Press, 1980. pp. 3-18. 17. Sindelar, W. F. and C. C. Kurman. Nitrosamine-indueed transplantable pancreatic ductal adenocarcinoma in inbred Syrian hamsters. J Nail Cancer lnst 67:1093-1100, 1981.
UEDA ET AL 18, Smith. P. A. and R. M. Case. Effects of cholera toxin on cychc adenosine 3',5'monophosphate concentration and secretory process in the exocrine pancreas. Bh~chml Btophvs At ta 277-290, 1975. 19. Suzuki, Y., S. Itagaki, N. Iwatsukl and N. Ueda. Secretory and electrical properties of synan hamster pancreas. J Ph.x ~lol S ~ Jpn 43: 275, 1981. 20. Takahashi. M., R. Runge, T. Donnelly and P. Pour The morphologic and biologic patterns of chemically induced pancreatic adenocarcinoma in Syrian golden hamsters after homologous transplantation. Cant er Lett 7: 127-133, 1979. 21. Townsend. C. M., Jr., R. B. Franklin, F B. Gelder. E. Glass and J. C. Thompson. Development of a transplantable model of pancreatic duct adenocarcinoma. Surgery 92: 72-78, 1982.
0196-9781/84$3 00 + O0
Electrophysiological Studies on the Cultured Cells Obtained From Transplantable Pancreatic Carcinoma in Syrian Golden Hamsters N. UEDA, Y. SUZUKI, M. UTSUMI, T. OBARA, K. OKAMURA AND M. NAMIKI Third D e p a r t m e n t o f Internal Medicine, A s a h i k a w a M e d i c a l College 3-11, 4-5, NishiAagura, A s a h i k a w a , J a p a n
UEDA, N., Y. SUZUKI, M. UTSUMI. T. OBARA, K. OKAMURA AND M. NAMIK1. Electrophysiological .~tudw.~on the t ultured cells obtained frmn transplantable pancreatw carcinoma in Syrian golden hamster~. PEPTIDES 5(2) 423-428, 1984.--A pancreatic ductal carcinoma was established as a transplantable tumor line in an inbred strain of Syrian golden hamsters. Intracellular recordings of membrane potentials and input resistance were made from cultured cells obtained from the transplanted tumors using indwelling glass microelectrode. The mean value of the resting membrane potential was -46.5_+ 1.8 mV (S.E.) In= 13), while the mean resting input resistance was 21.2±4.3 MI) (S.E.) (N=I3). Dibutyryl cyclic AMP f2 x 10-a M) caused a marked hyperpolarization of about 30 mV accompanied by a reduction of input resistance. The transplantable tumor and Its cultured cell line developed in this study have demonstrated their effectiveness as a reliable experimental model for use m pancreatic cancer research. Pancreatic ductal carcinoma Syrian golden hamster Transplantable pancreatic cancer Effect of secretin. VIP. and dibutyryl cyclic AMP Membrane potential Input resistance N-nltrosobts [2-hydroxypropyl] amine
P A N C R E A T I C cancer in humans has been on the increase during this century, taking an annual toll of about 8,000 in Japan. Moreover. because the disease is usually in its advanced stages upon discovery, there is a very low five-year survival rate [5]. A pancreatic ductal adenocarcinoma induced by N-nitrosobis (2-hydroxypropyl) amine (DHPN) in the Syrian golden hamster is remarkably similar to that occurring in man m its morphoioglc characteristics [4,12]. Therefore, the Syrian hamster is a useful model to study pancreatic cancer. Recently we succeeded in establishing a transplantable tumor line in an inbred strain of Syrian hamsters which appears to be suitable for use in further biological and therapeutic investigations. The present investigation was concerned with the bioelectrical properties of the cultured cells obtained from the tumor transplanted m the Syrian golden hamsters.
Following a period of progressive weight loss (between 30-40 weeks after initial treatment), the hamsters were killed by cervical dislocation. Pancreatic tumors were removed under sterile conditions and divided into two parts, one for transplantation and the other for histological examination.
Transplantation The tumor tissue was placed in a sterile petri dish with Hanks balanced salt solution. The tumor was minced into small pieces with scissors (about I mm cubes), and one or two fragments of the tumor tissue were inoculated subcutaneously with a trocar into the interscapular area of 4 to 6-week-old male syngeneic hamsters. Serial transplantations were performed in the same manner. Recipient hamsters were killed 6 weeks after the transplantation, and a part of the tumor tissue was transplanted serially.
Cell culture
METHOD
For the electrophysiological studies, a cell culture was made with a transplant from the twenty-fifth passage. The tumor was removed from a cutaneous incision, minced into fine fragments and incubated with RPMI 1640 medium containing 10% fetal calf serum and 0.1% collagenase (from Sigma) for l hr at 37°C with shaking.
TIIIllOI" hldllcfioll Eight-week-old male Syrian golden hamsters (Nisseiken Co.. Tokyo. Japan) were injected subcutaneously with DHPN in 0.9% NaCI solution at a dose of 250 mg/kg body wetght once weekly for 20 weeks.
423
424
UEDA ET AL.
FIG. I. Histoio~ fmdim~ of tm'hna_rytumor showins well dil~erendaled ~ t k
Individual cells and clump cells were passed through a free mesh, while coarse fibrous tissues and debris were remined in the mesh. The cells were washed three times with RPMI 1640 medium. The cell suspension (4× 10-s ceils/ml) was cultured for 1 week, in 95~ air and 5% CO, at 37~C. Measurement of Membrane Potential and Resistance
The culture plate (5 ml) was perfused with the standard Krebs-H_em~_it solution warmed to 37eC at a constant rate of 2.5 ml/min. The staadard solution had the following composition (raM): NaLl 103, KCI 4.7, CaLl 2.56, M41CI 1.13, NaHCOs 25, NaHPO( 1.15, D-glucose 2.8, Na pyruvate 4.9, Na 81uatmate 4.9, Na fitmarate 2.7. It was Iptased with 95% 02 5% CO~. Glass micmeiectro~s (1.5 mm in outer diameter) were pulled by a m k z o e l e c u ~ puller ( N ~ , Japan) and [i!kd with 3 M-KCI. The electrode had a tip resistance of about 15-20 MQ. Measurements of membrane potential and resistance were carried out by one intracelhflar microelectrode which was connected to an electrometer amplifier (W-P KS 700) allowins current ~ n and meamrement of membrane potential and resistance. Stimulants were added directly to the tissue bath close to the impaled microelectrode using a fine needle connected to a syrinlle. Sec~in and VII) were stored at -20*C and dissolved in a buffer solution just before application.
ductai cell carcinoma. (H and Ex 120.)
The drugs used were: VIP (Protein Research Foundation, Japan), secretin (Eisai, Japan) synthesized caendein (Kyowa Hakko, Japan), acetyicholine chloride (Wako chemicals, Japan), dibutyryl cyclic AMP (Sigma), cyclic AMP (Sigma). RESULTS Tumors were induced in all of the hamsters administered repeated injections of DHPN. Hiatoiogkal examinatioas of the original tumor showed typical features of welldifferentiated l x m c ~ duct adenocaeciaoma (Fig. I). The tran~lented tumors became psipsble at the inoculated site after 1 week and thereafter iprew ~ y . The tumors were suceessf~y passed through 30 Ileneratiorm, and the tnmspimttability rate was 67~ to 100~. The doublial; time of the tumor volume varied from 3.4 to 5.7 days in the 1lth to 18th generations, and the average doubling time was 4.1_.0.82 days. Histolollieal fmdialls of the transplaated tumor (25th generation) revealed it to be similar to those of the otis'hal tumor (Fig. 2). Culture cells of the transplanted tumor exhibited typical characteristics of an epithelial with larse nuclei, and the 1 or 2 small nu¢leoli were observed (Fig. 3). The mean resting membrane potential obtained from stable impalements comtinued for more than 3 mim,'tes was -46.$__.1.8 mV (S.E.) (n=13). The current-voltalle relationship was obtained by injecting rectanselar c.rref~s through
425
FIG. 2. Micro~cpic section from the twenty-fi/~h generation demonstrating the same h/stolosical appearance as the orig/nal tumor. (H and Exl20.)
FIG. 3. One week-old cultured cells. The cells are cuboidal and grow in sheets which are tightly adimrent to each other. (Phase contrast, original magnification x400.)
U E D A ET AL
426 VtP
MP(mv)
a KCI b
:
I I
i
1
mm
-~ 0 I -I° I -30
-2
-!
!025hA
-40
•
i
-$0
I 0 25 nA
i xlO-=A
-60 ,-';'0 50 ms
FIG. 5. The effect of VIP and KCI on the membrane potential and resistance. The top part shows a pen recording. 'vii) (0.35 V,g) (to obtain an estimated peak concentration of at least 2x 10-' M} did not cause any effect on the membrane potential and resistance, while an application of KCI (3.2 rag) (to achieve an estimated peak concentration of about 20 raM) caused an immediate depolarization. The lower part shows the time course ofelectrotonic potential change before and during the action of KCI.
-80 -90
I TOmV dbc~AP
I
a
m¢
~-] o b
¢ --
ii
i|
, , .... .
-4OmV
50ms
FIG. 4. The resting current-voltnge relattonship m pancreattc cancer cells. The lower parts shows membrane potential (taP) changes in response to recta,,tgular I~0 ms¢c current pulses of differcm magnitude. The top part shows plot of membrane potential as a function of magnitude and polarity of injected current.
]
I0
the recording electrode. The relation was almost linear between - 1 7 and - 8 6 mV (Fig. 4), which corresponds to an input resistance o f about 20 MI"L The mean resting input resistance within the range o f the linear current-voltage relation was 21.2_+4.3 Mfl (S.E.) ( n = 13). An application o f V1P (0.35 ~g) did not cause any c h a n p o f membrane potential and resistance, wh/le in tim same cell a direct addition o f KCI (3.2 rag) in order to augment K ion concentration resulted in a marked depolarization o f 45 mV accompanied by a slight reduction of input resistance (Fig. 5). In subscglutmt experiments, secretin (0.15 #g), acetylcholine (0.9 t~g), and caerulein (0.8 ng) had no effects on the membrane potential and resistance. Figure 6 shows the effect o f dibutyryl cyclic A M P (4 rag). Application o f dibutyryl cyclic A M P cammd a marked hyperlmlarization from - 3 3 mV to - 6 7 mV accompanied by the reduction of input resistance and shortening of time constant. The same results were obtained in 3 experiments. The mean membrane potential at the peak o f hyperpolarization induced by dibutyryl cyclic A M P was - 6 8 . 0 _+2.9 mV (S.E.) (n=3). tn contrast to the marked hyperpolarizing effect o f dibutyryl cyclic AMP, cyclic AMP, which is less lipophilic, caused a smaller hyperpolarization o f only 5 mV followed by a marked depolarization of 15 inV. DISCUSSION
The failure o f est',d~lishing on appropriate experimental ammal model for the study o f pancreatic cancer has resulted
, b
i
L
"
0 5nA
gnA
v ' -
F
e~ocal
FIG. 6. The effect of dibutyryl cyclic AMP (dbc AMP) on the membrane potential and resistance. The top part shows a pen recording Addition of dbc AMP (4 rag) to the culture plate to achieve an estimated peak concentration of at least 2×10 -~ M dunng intracelValar recordings caused m a r k e d hyperlmlarization accompanied by the reduction of input resistance. The lower part shows the time course of elcctrotonic p o t e n t i a l change before (a). during (b) and after (c) the action of dbc AMP.
in considerable delays in this field as compared to studtes on other gastrointestinal tract cancers. In 1974 Pour (,t (d. succeeded in inducing pancreatic duct adenocarcinoma in Syrian golden hamsters by chronic systemic adminhtlration of DHPN. Since this induced pancreatic cancer is quite similar morphologically to human ductal adenocarcinoma, their model appears to be suttable for use in the study o f pancreattc cancer {7, I I].
TRANSPLANTABLE PANCREATIC CANCER
427
Recently several reports on the establishment of transplantable tumor lines in inbred strains of Syrian hamsters have been published [6, 15, 17, 20, 21]. We also have succeeded in establishing a transplantable tumor line in our laboratory. The transplantable tumor demonstrated comparably fast growth and small possibility o f metastasis as well as histological similarity to human well-differentiated ductal adenocarcinoma. In addition, we confirmed that the histological findings of transplantable tumor are almost identical to those found in primary pancreatic cancer. These evidence support the usefulness o f this tumor in chemotherapeutic studies of pancreatic cancer as well as in biological and biochemical investigations. While morphological studies o f experimental pancreatic cancer have advanced, biological studies especially from the electrophysioiogical approach have not yet been conducted. In order to investigate the bioelectrical properties of pancreatic cancer cells, we have established a cell culture of transplantable tumor, and using these cells, have performed measurements o f membrane potentials and input reststance changes under stimuli of VIP, secretin, caerulein, acetylcholine, and dibutyryl cyclic AMP. The mean resting cell membrane potential was - 4 6 . 5 inV. This value is quite similar to already reported resting membrane potentials of several exocrine gland cells ( - 4 0 ~ - 7 0 mV) [9,19]. The current-voltage relationship was almost linear and exhibited a slight outward going rectification. This result indicated that the membrane conductance o f this cell is dominated by K + and C1-. As shown in Fig. 4, the increase o f K ÷ concentration in the perfusate caused a marked depolarization of the membrane potential, which further confirmed the essential role of K + ion in membrane conductance. Secretin and VIP, which are known as physiological stimulants o f the pancreatic ductal cell, had no effect on the membrane potenttai and input resistance in the cultured cancer cells in this study. Furthermore, potent stimulants of the pancreatic acinar cell such as acetylcholine and caerulein
also had no effect on them. However, admimstration of a penetrating analogue of cyclic A M P , which is thought to be an intraceilular mediator o f the effect of secretin and VIP on HCO..7 secretion in the pancreatic ductal cells, evoked a marked hyperpolarization accompanied by a reduction of input resistance [I, 2, 10, 16, 18]. Binding studies using J-Or'l-secretin in pancreatic cancer cells of hamsters and humans suggested the presence of secretin receptors on the membrane of these cells [3,15]. However, in our electrophysiological study, secretin or VIP had no apparent effect on the membrane potential or input resistance of the tumor cells, which indicates impaired membrane function incident to the neoplastic transformation. There is relatively little information about the effect of cyclic A M P on electrical properties of cell membranes. It has been demonstrated that glucagon evoked membrane hyperpolarization in liver parenchymal cells was mimicked by cyclic AMP, which is known as an intracellular mediator of the effect ofglucagon [8,14]. However, there is no available evidence on the effect of cyclic A M P on the electrical properties of tumor cell membranes. The effect of cyclic A M P will not, however, be finally solved until the effect o f secretin or VIP as well as cyclic A M P has been tested on an electrical properties o f those cells where the physiologically important receptors are probably localized (the centro-acinar or terminal duct cells). In conclusion, the transplantable tumor and its cultured cell line developed in this study have demonstrated their effectiveness as a reliable experimental model for use in pancreatic cancer research.
ACKNOWLEDGEMENT This work was supported by a Grant-in-Aid for Special Project Research from the Ministry of Education, Science and Culture. We thank Miss J. Watanabe for typing the manuscript and Miss C. Asai for technical assistance.
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