1022-7954/01/3709- $25.00 © 2001
Russian Journal of Genetics, Vol. 37, No. 9, 2001, pp. 1009–1013. Translated from Genetika, Vol. 37, No. 9, 2001, pp. 1207–1212.
Original Russian Text Copyright © 2001 by Zavadskaya, Zacharova, Kadulin, Kibardin, Kiselev, Gnuchev.
Angiogenesis is generation of new blood vessels.
This process hardly occurs in healthy adults but is
essential at certain stages of tumor progression and
metastatic spreading .
In oncological clinical practice, ectomy of the pri-
mary tumor in some cases results in rapid growth of
remote metastases. In other words, the primary tumor is
able to suppress the growth of metastases. The follow-
ing hypothesis has been advanced to explain this phe-
nomenon. The primary tumor has been assumed to
stimulate angiogenesis of its own vessels and to sup-
press angiogenesis in metastases and in secondary
tumors. Angiogenesis is a multifactorial process. The
primary tumor produces both activators and inhibitors
of angiogenesis, and the angiogenic phenotype is deter-
mined by their balance. However, the life time of inhib-
itors in circulation is longer than that of activators.
Hence, only inhibitors reach vessels of remote
metastases, and angiogenesis does not occur there. The
hypothesis has been experimentally conﬁrmed with
animal models .
Angiostatin, a 38-kDa protein that inhibits endothe-
lial proliferation, has been isolated among the ﬁrst fac-
tors blocking angiogenesis. This protein has been iso-
lated from serum and urine of mice carrying Lewis lung
carcinoma (LLC) and puriﬁed via chromatography
through several columns. Microsequencing has shown
that the amino acid sequence of angiostatin is homolo-
gous to a fragment of plasminogen . An endothelial
proliferation inhibitor produced by hemangiothelioma
(EOMA) cells has been similarly isolated and identi-
fied. This 20-kDa polypeptide (endostatin) proved to be
identical to the C-terminal fragment of collagen XVIII .
The proteins revealed have been studied with animal
models. It has been shown that regular injections of
angiostatin suppress the growth not only of LLC, but
also of at least three other mouse tumors even when
therapy starts after a tumor reaches 2% of the body
Even with mice, rather large amounts of the protein
are needed to obtain reliable data on therapy of various
tumors. Isolation from natural sources is difﬁcult and
low-efﬁcient. Recombinant endostatin overproduced in
has been reported to form an insoluble
precipitate . When obtained with the use of the bac-
ulovirus, recombinant endostatin speciﬁcally inhibits
proliferation of endothelial cells and is identical in
character of suppression to the natural protein. The
inhibition is sufﬁciently speciﬁc, as the growth of non-
endothelial, including EOMA, cells is not affected .
However, even with the baculovirus-based expression
system, the yield is too low for the protein to be used in
clinical practice for therapy of tumors and metastases.
As shown with animal models, recombinant
endostatin inhibits metastatic growth when injected at
g per mouse per day. Tumor growth inhibition is
53% at 25
g per mouse per day and 97% at 100
mouse per day; a dose of 200
g per mouse per day has
resulted in nearly complete regression of the primary
tumor in some animals .
Recombinant endostatin has also been used in ther-
apy of mice with transplanted melanoma B16F10, ﬁb-
rosarcoma E241, and hemangioma EOMA. All trans-
planted tumors rapidly regressed, and a toxic effect of
endostatin was not observed in treated mice .
Thus, endostatin, an inhibitor of endothelial cell
proliferation and angiogenesis, is promising for therapy
of various oncological disorders. However, the systems
employed in producing recombinant endostatin are
low-efﬁcient and fail to yield therapeutic doses of the
At present, the mammary gland of transgenic ani-
mals is considered as a promising system for produc-
Production of Recombinant Endostatin
in Milk of Transgenic Mice
E. S. Zavadskaya
, E. S. Zacharova
, S. G. Kadulin
, A. V. Kibardin
S. L. Kiselev
, and N. V. Gnuchev
Institute of Gene Biology, Russian Academy of Sciences, Moscow, 117334 Russia;
fax: (095)135-41-05; e-mail: email@example.com
Center of Medical Research, University of Oslo, Moscow, 117334 Russia
Received March 28, 2001
—Using the bovine
S1-casein gene, a genetic construct with an endostatin-coding fragment of the
mouse collagen XVIII cDNA was designed to express endostatin in milk of transgenic animals. Several trans-
genic mice were obtained. The mice secreted endostatin in milk at 70–300 ng/
l and transmitted this character
to their progeny.