Download Cellular Differentiation of Rat Uterine

[CANCER RESEARCH 35, 1713-1717, July 1975]
Cellular Differentiation of Rat Uterine Adenocarcinoma Cells by
Progesterone in Vitro
Souei Sekiya, Masaaki Kamiyama, and Hiroyoshi Takamizawa
Department of Obstetrics and Gynecology, Chiba University School of Medicine, 1-8-1 Inohana. Chiba 280, Japan
SUMMARY
Transplantable cloned HTP/C 1 culture was a stable line
derived from a rat uterine adenocarcinoma that was induced
by 7,12-dimethylbenz[a]anthracene
in vivo and did not
display density-dependent
inhibition of growth. This
HTP/C1 culture easily adapted to grow in a culture
medium containing progesterone, 8 ¿tg/ml.As compared
with HTP/C1 culture, HTP/C1/P8 culture grown in the
presence of progesterone was contact inhibited, and a
cellular differentiation was observed in the tumor tissues
that developed after inoculation of cells. These actions of
progesterone on uterine adenocarcinoma cells were com
pletely reversible on removal of the hormone in vitro.
These results appeared to indicate that progesterone was
involved in the regulation of both cellular proliferation and
differentiation. The possible mechanisms of the regulation
of rat uterine adenocarcinoma cells by progesterone are
discussed in relation to cellular levels of cyclic adenosine
3':5'-monophosphate in vitro.
Eagle's minimum essential medium supplemented with 10%
calf serum. Several biological characteristics of this cell line
in vitro were described previously (16).
Hormone. The stock solution of progesterone, 8 mg/ml
(Mochida Pharmaceutical Co. Ltd., Tokyo, Japan), was
prepared by dissolving it in ethanol and diluting it with
culture medium to give a final concentration of 8 jig/ml
before use. The solvent had no effect in the test system.
Transplantation of Cells. About IO6cells suspended in
0.2 ml of culture medium were injected s.c. into the
interscapular region of isologous newborn rats within 48 hr
after birth. The rats were sacrificed at 4 weeks after
inoculation of cells, and the sex, tumor weight, and
metastasis to the lung were examined. The tumor tissues
obtained were fixed in 10% neutral formaldehyde solution
and stained with hematoxylin and eosin, Van Gieson, and
silver staining method for histological examinations.
RESULTS
Isolation of Cells Adapted to Crow in the Culture Medium
Containing Progesterone. HTP/C1 culture easily adapted to
grow in the culture medium containing progesterone, 8
Recently, it has been reported that histological differenti
/ig/ml (HTP/C 1/P8 culture). However, higher concentra
ation of human endometrial adenocarcinoma was induced tions, more than 16 /ig/ml, seemed to be nonspecifically
by the administration of progestogens, irrespective of local toxic, and attempts to isolate cells that could grow in the
or systemic application of the drugs (1-3, 5-7,9-12, 20, 21).
culture medium containing progesterone at concentrations
We reported that progesterone at concentrations of 8 ¿tg/ml higher than 16 ¿tg/mlwere unsuccessful.
or higher was inhibitory to the growth of rat uterine
HTP/C 1 cells grew rapidly (doubling time, 22 hr) and
adenocarcinoma cells in vitro (17). However, the mech consisted of loosely adherent, elongated, less flattened cells
anisms of cellular differentiation by progesterone are not that showed a marked motility (Fig. 1). HTP/C 1/P8 cells,
known, particularly in cancer cells.
in contrast, were strongly adherent, polygonal cells with
This paper describes an attempt to evaluate the regula
characteristic granules in cytoplasms (Fig. 2). They prolifer
tory mechanisms of differentiation of rat uterine adenocar
ated slowly (doubling time, 32 hr) under the influence of
cinoma cells by progesterone in vitro.
progesterone. On the other hand, contact inhibition of cells
did not occur in HTP/C 1 culture, and cells began to pile up
after extensive growth on the surface. In contrast,
MATERIALS AND METHODS
HTP/C 1/P8 cells consisted of more flattened cells, and no
Cells. Transplantable cloned cell line HTP/C1, which overlapping or multilayering of cells was seen after division
was derived from the parent culture of a Sprague-Dawley ceased at confluence (Fig. 4).
The saturation density at confluency decreased from 17.4
rat uterine adenocarcinoma induced by intrauterine admin
istration of 7,12-dimethylbenz[a]anthracene in vivo, was x 10* cells/sq cm in HTP/C 1 culture to about 4 x 10«
used (16, 18). Cells were grown at 37°in a humidified 5% cells/sq cm in HTP/C 1/P8 culture 11 days after inocula
CO2 and cultivated in 60-mm Petri dishes (Falcon Plastics, tion of about IO5cells/plate.
On removal of progesterone and addition of fresh culture
Oxnard, Calif.). The culture medium consisted of modified
medium to HTP/C 1/P8 culture, the cells reverted to the
Received January 21, 1975; accepted March 26, 1975.
original morphology of cell colony within 5 days (Fig. 3)
INTRODUCTION
JULY 1975
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research.
1713
5. Sekiva et al.
shown in Table I, the average tumor weight after 4 weeks of
inoculation of IO8 HTP/C1/P8
cells was about one-half of
that after inoculation of HTP/C1 cells. In addition, the rate
of lung metastasis was significantly decreased in animals
given inoculations of HTP/C 1/P8 cells, as compared with
that produced by HTP/C 1 cells.
Histology of Tumor Tissues. The histológica! findings of
tumor tissues produced in animals given inoculations of
HTP/C 1 cells showed undifferentiated
adenocarcinoma
with large stromal reticulum fibers, and the glandular
structure was scarcely detected (Fig. 6). In contrast, histol
ogy of tumor tissues produced by HTP/C 1/P8 cells showed
a marked glandular structure with proliferation of stromal
tissues and confirmed a diagnosis of differentiated adeno
carcinoma (Fig. 7).
DISCUSSION
Fig. 1. HTP/CI
cell colonies 5 days after inoculation
oflO3 cells. Phase
contrast, x 100.
Fig. 2. HTP/CI/P8
cell colony grown in the culture medium contain
ing progesterone. 8 ¿ig/ml, at 5 days after the inoculation of IO3cells. Phase
contrast, x 100.
Fig. 3. HTP/CI/P8
cell colonies 5 days after removal of progesterone,
at 5 days after the inoculation of 10s cells. On removal of progesterone, the
cells reverted to the original morphology
100.
of cell colony. Phase contrast,
x
and to their original 3-dimensional growth within 11 days
(Fig. 5).
Tumorigenicity and Rate of Lung Metastasis of Cells. As
1714
Present results show that progesterone is involved in the
regulation of both cellular proliferation and differentiation
of rat uterine adenocarcinoma
cells. These actions of
progesterone on carcinoma cells are direct and reversible in
vitro.
Histológica! studies of human endometrial
adenocarcinomas secondary to the administration
of progestational
agents have been reported. Kistner et al. (9, 10) first
demonstrated that synthetic progestogens produced marked
changes, such as profound glandular atrophy and decidua!
conversion of the stroma in endometrial adenocarcinomas
following prolonged progestogen therapy. Verga and Henriksen (20) also noted that the structural changes consisted
of a more orderly and benign appearance of glandular
pattern, secretory distension of the acini, and extensive
acanthomatous
transformation.
The same observations as
above on the histological effects of progestational
agents
have been reported by other investigators (1 3, 5-7, 11, 12,
21). In addition to histological alterations, the cytological
changes in the cells of human endometrial adenocarcinomas
following progestogen therapy were observed to be: (a) a
decrease or cessation of mitotic activity, (b) pyknosis of the
nuclei, (c) less piling up of the cells and a tendency toward
single layering, and (d) increase in cytoplasm with promi
nent vacuoles, irrespective of local or systemic application
of the agents (1, 5, 6, 20, 21).
On the other hand, Nordqvist (13, 14) demonstrated that
human endometrial carcinomas showed a dose-dependent
sensitivity to progesterone in organ culture. Kohorn and
Tchao (11) also reported the effect of progesterone
on
endometrial carcinomas in vitro and suggested that proges
terone could directly produce differentiation of carcinoma
tissues, as reported in the normal human endometrium (4).
These results in vivo and in vitro suggest that progesta
tional agents directly increase differentiation of endometrial
carcinoma cells, as observed in the present study.
Although the biological role of cyclic AMP1 remains
•¿The
abbreviation
used is: cyclic AMP,
cyclic adenosine
3':5'-mono-
phosphate.
CANCER RESEARCH
VOL.35
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research.
Differentiation of Uterine Carcinoma by Progesterone
•¿
•¿
Fig. 4. HTP/C l/P8 culture at confluent state.
At 11days after the inoculation of IO5cells. In the
presence of progesterone, piling up of cells was
noticeably inhibited. Phase contrast, x 100.
Fig. 5. HTP/C I/P8 culture at confluent state
11 days after removal of progesterone, at 11days
after the inoculation of 10scells. Note piling up of
cells. Phase contrast, x 100.
•¿
,'
ffÉ,
VÕ&SL
/ «
¿> ,
obscure, it might involve the control of both cellular growth
and differentiation. This is supported by the observations
Suspension of 10*cells in 0.2 ml of culture medium were inoculated s.c.
that cyclic AMP levels are lower in transformed cells (15,
into interscapular region of isologous newborn rats. Only the tumors
19) and that agents that increase cellular cyclic AMP levels
produced in male rats were examined.
suppress the growth of cells (8). Therefore, whether proges
terone stimulates the formation of cyclic AMP levels within
ratswith
of
rat uterine adenocarcinoma cells remains to be elucidated
metas
wkafterwt/rat4
ofrats
and is currently under investigation. Cellular levels of cyclic
linesinoculatedHTP/CIHTP/CI/P8"No.
Cell
inoc
inoculationof
tasisthelung122
to
ulated1216Tumor (g)1.1
cells
AMP detected by radioimmunoassay are at least 3 times
higher
in HTP/C 1/P8 cells grown in the culture medium
±0.1"0.6
containing
progesterone, as compared to original HTP/C 1
±0.1No.
cells grown in the culture medium without hormone (S.
Sekiya, Y. Kikuchi, and H. Takamizawa, unpublished
" Mean ±S.E.
" HTP/C I cells growing in the culture medium containing progesterone.
data). As a result of the increase in cyclic AMP levels in
carcinoma cells by unknown mechanisms, the regulation
Table I
Tumorigenicity
of cells
JULY 1975
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research.
1715
5. Sekiya et al.
Fig. 6. Histological
picture of the tumor
tissues 4 weeks after inoculation of IO" HTP/CI
cells into isologous newborn rat. This reveals
undifferentialed
adenocarcinoma.
and a glandu
lar structure is scarce. H & E, x 100.
Fig. 7. Histological
picture of the tumor
tissues 4 weeks after inoculation
of 10*
HTP/C 1/P8 cells. Note
structure with proliferating
E, x 100.
a marked glandular
stromal tissues. H &
of both cellular growth and differentiation
might occur in rat uterine adenocarcinoma
by progesterone
cells.
ACKNOWLEDGMENTS
The authors greatly appreciate the histological examinations
Nagao. Department of Pathology, Chiba University.
by Dr. K.
REFERENCES
1. Anderson. D. G. Management of Advanced Endometrial Adenocar
cinoma with MedroxN progesterone Acetate. Am. Ì.
Obstet. Gynecol..
92: 87 99, 1965.
2. Bergsjö. P. Progesterone
and Progestational
Compounds
in the
Treatment of Advanced tndometrial
Carcinoma. Acta tndocrinol..
49:412 426, 1965.
1716
3. Bonté,}. B.. Drochmans,
A., and Ide, P. 6-a-Methvl-17
a-Hydroxy-
progesterone
Acetate as a Chemotherapeutic
Agent in Adenocar
cinoma of the Uterus. Acta Obstet. Gynecol. Scand., 45: 121-129,
1966.
4. Csermely. T.. Demers, L. M., and Hughes, E. C. Organ Culture of
Human Endometrium.
Effect of Progesterone. Obstet. Gynecol., 34:
252 259, 1969.
5. Hudson, C. N., Chir. M.. and Stansfeld, A. G. The Histological
Changes Produced in Untreated Adeno-Carcinoma
of the Endome
trium by a Progestogen. }. Obstet. Gynaecol. Brit. Commonwealth.
74:442 444, 1967.
6. Hustin, }. Endometrial Carcinoma and Synthetic Progestogens Re
sults of Intrauterine Treatment. }. Obstet. Gynaecol. Brit. Common
wealth, 77:915-921.
1970.
7. John, A. H.. Cornes, J. S., Jackson, W. D., and Bye, P. Effect of
Systemically Administered Progestogen on Histopathology
of Endo
metrial Carcinoma. J. Obstet. Gynaecol. Brit. Commonwealth,
81:
786 790, 1974.
CANCER RESEARCH
VOL. 35
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research.
Differentiation of Uterine Carcinoma by Progesterone
8. Johnson, G. S., and Pastan, I. Change in Growth and Morphology of
Fibrohlusls by Prostaglandins.
J. Nati. Cancer Inst., 47: 1357 1364,
1971.
9. Kistner. R. W. Histológica! Effects of Progestins on Hyperplasia and
Carcinoma In Situ of Endometrium.
Cancer. 12: 1106 I \22. 1959.
10. Kistner, R. W., Griffiths. C. T.. and Craig, J. Use of Progestational
Agents in the Management
of Endometrial
Cancer. Cancer, 18:
1563 1579. 1965.
11. Kohorn. E. I., and Tchao, R. The Effect of Hormones on Endometrial
Carcinoma in Organ Culture. J. Obstet. Gynaecol. Brit. Common
wealth, 75: 1262-1267, 1968.
12. Moe, N. Short-Term
Progestogen Treatment of Endometrial Carci
noma. Acta Obstet. Gynecol. Scand., 51: 55 62, 1972.
13. Nordqvist. S. Hormone Effects on Carcinoma of the Human Uterine
Body Studied in Organ Culture. A Preliminary Report. Acta Obstet.
Gynecol. Scand.. 43: 296 307. 1964.
14. Nordqvist. S. The Synthesis of DNA and RNA in Human Carcinomatous Endometrium
in Short-Term
Incubation
In Vitro and Its
Response to Oestradiol and Progesterone. J. Endocrinol.. 48: 29 38.
1970.
15. Otten, J.. Johnson. G. S.. and Pastan. I. Cyclic AMP Levels in
Fibrohlasts: Relationship to Growth Rate and Contact Inhibition of
JULY
Growth. Biochem. Biophys. Res. Commun.. 44: 1192 1198, 1971.
16. Sekiya, S., Kikuchi, Y., and Takamizawa. H. High- and Low-Tumorigenic Culture Lines of Rat Uterine Adenocarcinoma
and Their
Isozyme Patterns of Láclate Dehydrogenase and Hexokinase. Cancer
Res., 33: 3324 3329, 1973.
17. Sekiya. S.. Kikuchi. Y.. and Takami/awa.
H. The Effects of Hor
mones and Chemotherapeutic
Agents on Rat Uterine Adenocar
cinoma Cells in Tissue Culture. Am. J. Obstet. Gynecol.. 119:
675-680, 1974.
18. Sekiya. S.. Takami/awa.
H.. Wang, F., Takane, T., and Kuwata. T. In
Vivo and In Vitro Studies on Uterine Adenocarcinoma
of Rat Induced
by 7,l2-Dimcthylben/[a]anthracene.
Am. J. Obstet. Gynecol., 113:
691 695. 1972.
19. Sheppard. J. R. Difference
in the Cyclic Adenosine
3'.5'-Monophos-
phate Levels in Normal and Transformed
Cells. Nature New Biol.,
236: 14 16. 1972.
20. Verga. A., and Henriksen, E. Histologie Observations on the Effect of
l7-a-Hvdroxyprogeslerone-l7-jV-Caproale
on Endometrial
Carci
noma. Obstet. Gynecol.. 26: 656 664, 1965.
21. Went/. W. B. Effect of a Progestational
Agent on Endometrial
Hyperplasia and Endometrial Cancer. Obstet. Gynecol., 24: 370 375,
1964.
1975
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research.
1717
Cellular Differentiation of Rat Uterine Adenocarcinoma Cells by
Progesterone in Vitro
Souei Sekiya, Masaaki Kamiyama and Hiroyoshi Takamizawa
Cancer Res 1975;35:1713-1717.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/35/7/1713
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research.