Full text of DOI:10.1007/BF02900628

1
Materials and methods
Cloning of aminopeptidase N
promoter and its activity in
hematopoietic cell and differ-
ent tumor cell lines
(ν) Cell cultures. Myeloblatic cell line KG1a (gift
from Prof. Zheng Dexian, Institute of Basic Medical Sci-
ences, Peking Union Medical College and Chinese
Academy of Medical Sciences) was cultured in Isocove’s
modified Dulbecco’s medium (Gibco). T lymphocyte cell
line Jurkat (also kindly provided by Prof. Zheng Dexian),
lung adenoma cell line Anip973, and hepatoma cell line
BEL 7402 were grown in RPMI 1640 medium (Hyclone).
Tong cancer cell line TCA and hepatonoma cell line
HepG2 were maintained in Dulbecco’s modified Eagle
medium / F12 medium (Sigma). Esophageal cancer cell
line NEC (gift from Prof. Lu Shixin, Cancer Institute,
Peking Union Medical College and Chinese Academy of
Medical Sciences) was cultured in M199 medium (Gibco).
Each medium supplemented with 10% fetal bovine serum
and 100 U/mL penicillin plus 100 U/mL streptomycin.
LIANG Libo1, MA Yewei 1, ZHAO Qingzheng1,
YANG Jun2, LIU Yuying1 & WANG Zheng1
1. Cancer Institute, Peking Union Medical College and Chinese Acad-
emy of Medical Sciences, Beijing 100021, China;
2. Institute of Blood Transfusion Medicine, Beijing 100850, China
Correspondence should be addressed to Zhao Qingzheng
Abstract
Aminopeptidase N (APN) promoter region was
cloned and sequenced from peripheral blood mononuclear
cells. The recombinant reporter construct containing the
promoter and luciferase gene, designated pXP1-APNLuc,
was introduced into myeloblastic cell line, T lymphocyte cell
line and various tumor cell lines. Luciferase assay showed
that APN upstream promoter is myeloid-specific for high
expression in myeloblastic cell line and much lower expres-
sion in T lymphocyte cell line. The promoter activity was
relatively high in lung adenoma cell line compared with
other tumor cell lines including hepatoma cell line, tong can-
cer cell line and esophageal cancer cell line in which the
promoter activity significantly diminished or was almost
undetectable. The characteristics of APN promoter may pro-
vide a new strategy for specific myeloprotection while tumor
patients are being treated with chemotherapy and/or radio-
The cells were incubated at 37ć in a humidified atmos-
phere of 5% CO2.
(ξ) Amplification of APN promoter. Peripheral
blood mononuclear cells were isolated by Ficoll-Hypaque
density centrifugation. One Pg of DNA purified from the
cells was used for PCR. After hot start at 95ć for 5 min,
thirty cycles of PCR were performed: denaturation at
95ć for 1 min, annealing and extension at 68ć for
3.5 min, then the extension completed at 68ć for 7 min.
The primers were 5ĄGATGGATCCTTCTGAACTAGG-
AGT3Ąand 5ĄTATGAATTCGCTGGAACCTGGACCC-
therapy.
Keywords: aminopeptidase N, myeloid-specific promoter, reporter
gene.
T3Ąin which a BamHĉ site and an EcoRĉ site (un-
derlined) were added. The reaction system contained 5%
DMSO.
Chemotherapy and radiotherapy are two of the
mainstays of current synthetic therapy for tumor patients.
However, myelosuppression induced by anticancer agents
and irradiation is one of the major side effects that limit
the effectiveness of the treatments. Introduction of drug
resistance genes and irradiation resistance genes into he-
matopoietic precursors leading to protection against the
hematotoxicity has been proposed to reduce myelosup-
pression[1,2]. The administration of the genes is capable of
protecting not only hematopoietic cells but also the tumor
cells invaded into bone marrow since the expression of the
exogenous genes is not specific. One of the solutions to
this problem is to develop vectors containing specific
promoter elements that regulate the expression of drug- or
radiation-resistance genes only in hematopoietic cells.
The expression of aminopeptidase N (APN) is highly
lineage-specific in the earliest myeloid progenitor cells
and the derived differentiated cells[3]. In the study we
cloned the promoter of APN gene and assayed the activity
in myeloblastic cell line and different kinds of tumor cell
lines.
(ο) Plasmids construction. The APN promoter
fragment amplified by PCR was cloned into the BamHĉ
and EcoRĉ sites in the pBlueScript Ċ SK (Stratagene)
to generate a construct named pBSAPN. The plamid was
then digested with BamHĉand Hind ċ, the released
insert was ligated into pXP1 (gift from Prof. Paul Dobner,
University of Massachusetts) to generate the pXP1- AP-
NLuc construct. The cloned fragment was sequenced by
the dideoxynucleotide chain termination method. Se-
quencing primers were T7 and T3 that were complemen-
tary to sites flanking the multiple cloning sites in the vec-
tor pBlueScript Ċ SK.
(π) Reporter gene analysis. Transfection was per-
formed with 6 Pg of the reporter plasmid pXP1-APNLuc
and 2 Pg of E-gal control plasmid PCH110 (Pharmacia).
KG1a and Jurkat cell lines were electroporated with Gene
Pulser (BIO-RAD) set at 960 PF and 200 V. BEL7402,
HepG2, TCA and NEC cell lines were transfected by
DEMRIE-C Reagent (Gibco). Assays to detect luciferase
and E-gal activity were performed as described[4]. Prepa-
ration of cell extracts and analysis of luciferae and E-gal
Chinese Science Bulletin Vol. 46 No. 19 October 2001
1649

NOTES
activities were carried out using luciferase assay system
and E-galactosidase enzyme assay system (Promega) ac-
cording to the recommended procedures of the manufac-
turer. Relative luciferase activity = luciferase activ-
ity/E-gal activity.
were produced. After the digestion with BamHĉ and
Hind ċ , a 0.52 kb insert was also released from
pXP1-APNLuc. The orientation of the promoter was con-
firmed by sequencing pBSAPN (fig. 2). These data dem-
onstrated that the size and the orientation of the promoter
fragment were correctly cloned in the vector.
2
Results
(ο) DNA sequencing. The promoter fragment was
sequenced with T3 and T7 primers and the sequences
from five clones were identical, indicating that the substi-
tutions (see below) in the promoter sequence were not
created by the PCR procedure. Compared with the results
of Shapiro[5], two differences were detected: a AoG sub-
stitution at –192 and a GoA substitution at –82. The dif-
ferences may represent the polymorphism in APN pro-
moter.
(ν) Amplification of APN promoter. It was very
difficult to amplify APN promoter because the region was
GC rich. In order to destroy the secondary structure of
DNA and lower the annealing temperature, DMSO was
added to the reaction system and a two-step procedure of
PCR was carried out. A single band of 520 bp was pro-
duced (fig. 1).
(π) Reporter gene assay. The reporter gene was ex-
pressed at highest level in myeloblatic cell line KG1a and
significantly decreased in T lymphocyte cell line Jurkat
and other tumor cell lines. The relative luciferase activity
in BEL7402, HepG2, Jurkat, NEC, TCA, 44.5% and
Anip973 accounted for 3.9%, 3.5%, 15.9%, 40.5% and
83.1% of the activity in KG1a, respectively (fig. 3).
3
Discussion
APN is a member of the family of membrane-bound
metallopeptidases. APN gene is encoded by 20 exons that
extend more than 30 kb on long arm of human chromo-
some 15[6]. Two physically distinct promoters, 8 kb apart
from each other, are located on APN genomic DNA. APN
driven by upstream promoter can be detected on the earli-
est progenitor cells committed to the granulocyte/mono-
cyte developmental pathway as well as its more different-
Fig. 1. Amplification of APN promoter. 1, 100 bp DNA marker; 2,
5%DMSO; 3, 2.5%DMSO; 4, ꢀ%DMSO; 5, O/Hind ċ DNA marker.
Fig. 2. Vectors construction and identification. (a) pBSAPN identifica-
tion. 1, BamHĉ/EcoRĉ digests pBSAPN; 2, O/Hind ċ DNA marker.
(b) pXP1-APNLuc indentification. 1, BamHĉ/Hind ċ digests pXP1-
APNLuc; 2, O/Hind ċ DNA marker.
Fig. 3. Activity of APN promoter in myeloblastic cell line KG1a and
various tumor cell lines.
tiated progeny, but is not found on normal hematopoietic
of other lineages or their progenitors while downstream
promoter acts in liver, small intestine and endometri-
um[7—9]. The sequence within upstream promoter contains
elements that regulate myeloid-specific gene expression.
(ξ) Vector construction and identification. The
resulting construct pBSAPN was cut with BamHĉ and
EcoRĉ and a 0.52 kb fragment and a 2.9 kb fragment
1650
Chinese Science Bulletin Vol. 46 No. 19 October 2001

References
The genomic fragment extending from –411 to –291 con-
tains a Myb binding site and three Ets binding sites that
would result in a significant reduction of the promoter
activity if mutated or deleted. In this study, APN myeloid
promoter is cloned by PCR and the sequence shows that
the fragment of – 411— ꢁ200 which is highly associated
with myeloid-specific expression is conservative, in
agreement with the results of Shapiro although the poly-
morphism is present in the promoter region[5]. Reporter
gene analysis confirms that the cloned promoter sequence
is myeloid-specific for the high level of activity in mye-
loblastic cell line KG1a and much lower level of activity
in T lymphocyte cell line Jurkat. The results of transient
transfection also revealed that the promoter activity re-
duced significantly in tong cancer cell line, esophageal
carcinoma cell line and especially in hepatoma cell lines.
But the promoter activity is relatively high in lung ade-
noma cell line compared with other tumor cell lines tested.
The utilization of upstream promoter in lung may explain
1. Yang, J., Wu, Y., Ma, P. et al., Construction and expression of ret-
roviruses encoding dual drug resistance genes in human umbilical
cord blood CD34+ cells, Chinese Science Bulltin, 2000, 45: 372.
2. Greenberger, J., Gene therapy for radiation protection, Gene Ther.,
1999, 1495.
3. Shapiro, L.H., Myb and Ets proteins cooperate to transactivate an
early myeloid gene, J. Biol. Chem., 1995, 270: 8763.
4. Dong, Z.Z., Wang, X.F., Zhao, Q.Z. et al., DNA methylation con-
tributes to expression of the human neurotensin/neuromedin N
gene, Am. J. Phsiol., 1998, 274: G535.
5. Shapiro, L.H., Ashmun, R.A., Roberts, W.M. et al., Separate pro-
moters control transcription of the human aminopeptidase N gene
in myeloid and intestinal epithelial cells, J. Biol. Chem., 1991,
266: 11999.
6. Lerche, C., Vogel, L.K., Shapiro, L.H. et al., Human aminopepti-
dase N is encoded by 20 exons, Mamm. Genome, 1996, 7: 712.
7. Olsen, J., Lausten L, Troelsen, J., HNF1D activates the aminopep-
tidase N promoter in intestinal (Caco-2) cells, F.E.B.S. Lett., 1994,
342: 325.
8. Kataoka, H., Joh, T., Miura, Y. et al., AT motif binding factor 1-A
(ATBF1-A) negatively regulates transcription of the aminopepti-
dase N gene in the crypt-villus axis of small intestine, Biochem.
Biophys. Res. Commun., 2000, 267: 91.
this observed phenomenon[10,11]
.
In the treatments of breast carcinoma, ovarian carci-
noma and meloma, introduction of drug-resistance genes
and irradiation-resistance genes into myeloid cells is an
effective approach for preventing myelosuppres-
sion[2,12—14]. However, there are some limits: the nonspe-
cific expression of exogenous genes also results in in-
creased drug and radiation resistance of tumor cells, thus,
this nonspecific approach can only be used for the patients
whose tumor cells have not invaded into bone marrow.
Clinically, most of tumor patients at diagnosis are at the
middle or later stage and tumor cells have already invaded
into bone marrow, which are the obstacles of wide clinical
use. Application of APN myeloid-specific promoter lead-
ing to high expression of drug-resistance genes and irra-
diation-resistance genes in hematopoietic cells and low or
not expression in tumor cells would overcome the limits
and play an important role in the future gene therapy for
the patients with hepatoma and other tumors.
9. Olsen, J., Classen-Linke, I., Sjostrom, H. et al, Pseudopregnancy
induces the expression of hepatocyte nuclear facor-1E and its tar-
get gene aminopeptidase N in rabbit endometrium via the epithe-
lial promoter, Biochem. J., 1995, 312: 31.
10. Gillis, L.D., Pendley, D.S., Funkhouser, J.D., The major APN
transcript of the alveolar type II epithelial cell originates from a
unique upstream promoter region, B.B.A., 1998, 1399: 126.
11. Jiang, X., Tangada, S., Peterson, R.D.A. et al., Expression of
aminopeptidase N in fetal rat lung during development, Am. J.
Phsiol., 1992, 263: L460.
12. Deisseroth, A.B., Holmes, F., Hortobagyi, G. et al., Use of
safety-modified retrovirus to introduce chemotherapy resistance
sequences into normal hematopoietic cells for chemoprotection
during the therapy of breast cancer: a pilot trial, Hum. Gene Ther.,
1996, 7: 401.
13. Kavanagh, I., Hanania, E., Giles, R. et al., Genetic modification of
cells used for transplant following intensive therapy for ovarian
cancer and breast cancer, Blood, 1996, 88(Suppl 1): 272a.
14. Suresh, A., Tung, F., Moreb, J. et al., Role of manganese superox-
ide dismutase in radioprotection using gene transfer studies, Can-
Acknowledgements The authors thank Prof. Lu Shixin for his gift of
NEC cell line and Prof. Zheng Dexian for his gift of KG1a and Jurkat
cell line. This work was supported by the National Natural Science
Foundation of China (Grant No. 39970818).
cer Gene Ther., 1994, 1: 85.
(Received May 18, 2001)
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