1. No category

From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
1994 84: 467-473
Progesterone-associated endometrial protein--a constitutive marker
of human erythroid precursors
M Kamarainen, L Riittinen, M Seppala, A Palotie and LC Andersson
Updated information and services can be found at:
http://www.bloodjournal.org/content/84/2/467.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
Progesterone-Associated Endometrial Protein-A Constitutive Marker of
Human Erythroid Precursors
By Meerit Kamarainen, Leena Riittinen, Markku Seppala, Aarno Palotie, and Leif C. Anderson
Progesterone-associated endometrial protein (PAEP/PP14)
is a 28-kD glycoproteinwith sequence homology to p-laaoglobulins containing a retinol-binding motif. PAEP/PP14 is
present at nanomolar concentrations in human serum.It is
producedbysecretoryanddecidualizedendometriumin
women and by seminal vesicle epithelium
in men. We report
here that PAEP mRNA is constitutively expressedin normal
hematopoietictissue.Westernimmunoblotting
ofbone
marrow cells with rabbit antibodiesto PAEP gave a bandat
28 kD, and immunocytochemical staining with monoclonal
antibodies localized PAEP into the cytoplasm of erythroid
precursors representing different stagesof the normoblast
series.PAEP was not detected in mature red Mood cells,
platelets, or in cells of the mydoid lineage. UntreatedK562
leukemia cells did not contain PAEP, whereas treatment of
the cells with tetradecsnoylphorbol acetate (TPA) induced
of the intact
strong expression of PAEP mRNA and synthesis
protein that was found both in the cytoplasm of the differentiating cells and in the supernatant of TPA-treated cultures. These findings add a new member to the growing
family of genesthat are constitutively expressed both
in the
reproductive tract andin the hematopoietic system.
0 7994 by The American Society of Hematology.
H
mined from May-Griinwald Giemsa-stained cytofuge slides. In the
samples used here, the proportion of morphologically distinguished
erythroid precursors was around 10% of the total cell population.
Purified platelets were obtained from the Finnish Red Cross Blood
Transfusion Service (Helsinki, Finland).
Cell culture and induction of cell differentiation. The hematopoietic cell lines K562, U937, and HL-60 were purchased from the
American Type Culture Collection (Rockville, MD). The human
megakaryoblastic cell lines Dami and HPRC were kindly provided
by Dr Riitta Alitalo (Transplantation Laboratory, University of Helsinki). The cells were cultured in RF"I 1640 medium (GIBCO,
Grand Island, NY) supplemented with 10%fetal calf serum (Flow
Laboratories, Strathclyde, UK) at 37°C in a humidified atmosphere
of 5% CO, in air. To induce differentiation, K562, U937, and HL60 cells were cultured in the presence of lo-' m o m tetradecanoylphorbol acetate (TPA), lo-' m o m RA, or 2 mmoVL sodium butyrate
for 3
ImmunoJuorometric assay. To measure the PAEP production,
K562 cells were cultured with or without 8 X lo-' m o m TPA
for 24, 48, 72, 96, or 120 hours, and PAEP immunoreactivity was
quantitated from the medium byan immunofluorometricassay essentially as described previously'* using monoclonal antibodies
(MoAbs)" produced against purified PAEP/PP14.20Antibody-containing ascitic fluids were purified using protein G-sepharose (MoAb
Trap G; Pharmacia LKB Biotechnology Inc. Piscataway, NJ).
MoAbs F23-9G2 were labeled with DELFIA Eu-labelling reagent
(Eu-chelate of isothiocyanatobenzyl-diethylene-triaminetetra acetic
acid, Wallac Ltd. Turku, Finland) and used in theassay at a concentration of 50 ng/2C!O &/well. MoAbs F25-9D8 were used for solidphase coating (2 pg/200 &/well). In the assay, standards (pooled
UMAN PROGESTERONE-associated endometrial protein (PAEPPP14) is a 28-kD glycoprotein synthesized
by secretory and decidualized endometrium.ls2Studies by in
situ hybridization histochemistry have localized the expression of PAEPPP14 mRNA in the glandular epithelial cells
of endometrium and decidua sp~ngiosa.~"
This protein has
also been found in the epithelium of seminal vesicles and it
is secreted into the seminal plasma? PAEP was previously
known as placental protein 14 (PP14), progestagen-dependent endometrial protein (PEP), and pregnancy-associated
endometrial a2-globulin (a2
-PEG)."8 Human Gene Mapping
Committee has suggested the name PAEP for this protein.'
PAEPPP14 has significant structural homology to &lactoglobulins of various species." This class of proteins also
contains the retinoic acid (RA) binding motif." The N-terminus of PAEPPP14 has 22% sequence homology to human
retinol binding protein.12
The biologic functions of PAEP are still enigmatic. Recent
findings indicate that PAEP has immunoregulatory activities.
The addition of PAEPPP14 suppresses proliferation of lymphocytes stimulated in vitro by mitogens or by allogeneic
cells in mixed lymphocyte c ~ l t u r e s . l ~Okamoto
,'~
and cow o r k e r ~ have
' ~ found that the cytolytic activity of natural
killer (NK) cells is inhibited in the presence of purified
PAEPPP14.
In this study, we show that precursors of the erythroid
lineage in normal human bone marrow (BM) constitutively
express PAEP protein. The human erythroleukemia cell line
K562 also expresses PAEP mRNA and protein after induced
differentiation in vitro.
MATERIALS AND METHODS
Tissues. Endometrium was collected after informed consent
from women undergoing hysterectomy for medical reasons (fibroids). The study was approved by the Ethical Committee of the
Department of Obstetrics and Gynecology, Helsinki University Central Hospital. After removal of the representative tissues for routine
histopathologic examination the remaining tissues were frozen in
liquid nitrogen. Samples from secretory phase endometrium were
used for this study.
Hematopoietic cells. Normal BM was obtained from diagnostic
aspirates or recovered from pieces of ribs normally resected during
thoracic surgery. The mature red blood cells (RBCs) were lysed
in 0.84% ammonium chloride andused for the analysis without
fractionation. The cell composition of the final sample was deterBlood, Vol 84, No 2 (July 15). 1994: pp 467-473
From Departments I and I1 of Obstetrics and Gynecology, Department of Clinical Chemistry, and Department of Pathology, University of Helsinki, Finland.
Submitted June 3, 1993; accepted March 23, 1994.
Supported by grants from the Sigrid Jusdlius Foundation, the
Academy of Finland, the Finnish Cancer Society, and the University
of Helsinki.
Address reprint requests to Meerit Kdmilrdinen, MSc, Research
Laboratory, Departments I and I1 of Obstetrics and Gynecology,
University of Helsinki, Haartmninkatu 2, SF-00290 Helsinki, Finland.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
OOO6-4971/94/8402-0031$3.00/0
467
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
KAMARiilNEN ET AL
468
A B C
-
D
Fig l. Amplification ofPAEPcDNAbyPCRin
human B M cells.
Upper panel: one fifth of the amplified reaction was applied to 1.5%
agarose gel and stainedwith ethidium bromide. Lower panel: Southern blot analysis of the gel. A shows human B M cells. B shows negative control representing first-strand cDNA synthesis lacking reverse
transcriptase. C shows negative control representing amplification
of all the chemical components without RNA. D shows positive control that represents amplification of TPA-treated K562 cell cDNA.
pregnancy serum containing measured PAEP concentrations) or
samples werefirstincubated overnight at4°C. After washing, the
wells were incubated with labeled MoAbs for 2 hours and further
treated as described before.Is The detection limit of the assay (1.5
pgIL) was defined as the PAEP concentration corresponding to the
mean fluorescence signal of 20 replicates of a zero sample plus 2
SD. The measuring range we used was 3.1 to 400 &L. The intraassay variation (n = 10) was 4.4% at the level of 105 p g k , 8.2%
at the level of 21 pg/L and 25% at the level of5 pgL. The interassay
variations ( n = I 1 ) were 4.7%. IS% and 18%. respectively.
/tnn?rorost~~inb?g.~.
Cytocentrifuged smears of BM cells were immunostained using 1300 diluted anti-PAEP MoAbs (clone 105
DHIFI) in an indirect peroxidase method" and counterstained with
hematoxylin to allow morphologic identification ofthe cells. For
double immunofluorescence. smears ofBM cells were stained with
1:50 diluted anti-PAEP MoAbs together with 1:100 diluted rabbit
antiglycophorin A antibodies,I6 followed by affinity purified, fluorescein isothiocyanate (F1TC)-conjugatedgoat antimouse Ig and tetramethylrhodamine isothiocyanate (TR1TC)-conjugated goat antirabbit Ig (Dako AIS. Denmark). Cytocentrifuged smears of K562,
U937, and HL-60 cells before and after induced differentiation and
the megakaryoblastic cell lines Dami and HPRC were fixed for 20
minutes in 3.5% paraformaldehyde, permeabilized with 0.1% NP40 in phosphate-buffered saline (PBS) for 10 minutes and stained
with anti-PAEP MoAbs. followed by affinity-purified, FITC-conjugated goat antimouse Ig.
M e t d d i c lcrheling und irnrllto?oI~rccipitation. K562 cells were
incubated for 2 hours in methionine-free Eagle's minimum essential
medium (GIBCO, Paisley, UK) before labeling for 2 hours at 37°C
with 0.2 mCiImL %methionine. The cells were collected, washed
twice in PBS and lysed in immunoprecipitation buffer (10 mmol/L
TRIS; pH 7.5. 50 mmol/L NaCI. 0.5% sodium deoxycholate, 0.5%
Nonidet P-40,0.l% sodium dodecyl sulfate (SDS), 0. I U of aprotinin
per mL). Three microliters of mouse ascitic fluid containing antiPAEP MoAbs (clone 105 DHI FI ) was added to the lysates, and
immunoprecipitations were performed usingprotein A-Sepharose
with and without the addition of 10 pg purified PP14 protein.''' The
samples were separated by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) on 12% gels. The radioactive proteins were visualized
by autoradiography.
Western inlmltnob/otting. Lysates of BM cells were immunoprecipitated with polyclonal rabbit anti-PAEP antibodies. The precipitates were separated under nonreducing conditions on SDS-PAGE,
transferred to nitrocellulose membranes and blotted with polyclonal
rabbit anti-PAEP antibodies,"' followed by"'I-labcled swine antirabbit Ig (Amersham International plc, Amersham, UK). The blots
were developed by autoradiography. Western blots of lysates of
K562 cells, before and after induced differentiation as well as of
lysates of platelets. were visualized by enhanced chemiluminiscence
according to the manufacturer's instructions (Amersham).
Northern blot nncl1ysi.s. RNA was isolated fromwhole cells by
the guanidinium isothiocyanate extraction method. Poly(A)+RNA
was isolated using oligo(dT)-cellulose. Samples (5 pg) were denatured with 6.3% formaldehyde and 50% formamide. electrophoresed.
and transferred tonylon membranes. The filterswere hybridized
with"P-labeledPP14
cDNA"' generously provided byDr Mervi
Julkunen (Departments 1 and I1 of Obstetrics and Gynecology. University of Helsinki). Labeling was performed by the randomoligonucleotide priming technique, using a Multiprime DNA labeling systems kit (Amersham). For autoradiography, Kodak XAR film
(Eastman-Kodak, Rochester, N Y ) was exposed to the filtersfor I and
14 days at -70°C. Rat glycaraldehyde-%phosphatedehydrogenase
(GAPDH) cDNA probe was used toquantify the amount and quality
of loaded Poly(A)+RNA.
In situ h.vbric/izotion. Cytocentrifuged smcars of BM cells were
fixed in 4% paraformaldehyde and stored at 4°C in PBS until used.
"S-labeled PAEP cRNA probes were generated by digestion of a
pGEM-3 blue vector containing PP14 cDNA'" in sense and antisense
orientation with S I / 1 restriction endonuclease, followed by in vitro
transcription with T7 RNA polymerase. A pGEM-3 blue vector was
linearized by Xtnn I restriction endonuclease to allow synthesis of
Fig 2. PAEPmRNA expression in B M cells. In situ hybridization
was performed with antisense (A) and sense (B1 PAEP probes.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
PAEP IN HUMAN ERYTHROIDPRECURSORS
469
I*
Fig 3. PAP-immunostaining with anti-PAEP MoAbs of B M cells, counterstained with hematoxylin and photographed (AI at 200 X and (B)
at 1,000 x magnification using a green filter t o visualize the reaction product. C shows Western immunoblot analysis of bone marrow cells.
anti-PAEP antibodies of immunoprecipitates obtained
Lane a shows patternsof standard proteins. Lane b shows pattern obtained with rabbit
from lysatesof B M cells using rabbitanti-PAEP antibodies. The positionof PAEP is indicated by arrow. Lane C shows control with nonimmune
serum. D shows immunofluorescense staining forPAEP with anti-PAEP MoAbs followed by affinity-purified, FITC-conjugated goat-antimouse
lg. E shows imrnunofluorescense staining with rabbit-antiglycophorin
A antibodies followedby affinity-purified TRITC-conjugated goat-antirabbit lg. IF) shows the corresponding phase-contrast microscopic presentation.
a "S-laheled nnnsenseRNAproheusingSP6RNApolymerase.
Nonsense and PAEP sense strands were used for control o f nonspecilic hinding. Pretreatment of slides. prehyhridization and hyhridization were performed using previously described protocols:' Briel-ly.
the cells were pretreated with proteinase K ( I pg/nlL). 0. I mollL
triethanolamine.and
0.25% aceticanhydride
i n triethnnolamine
buffer. Prehybridization was performed at ronnl temperature for 2
hours in a buffer composed of 50% formamide. 0.6 mollL NaCI. I O
mmollLTRIS-HCI, pH 8.0. 20 mmol/Ldithiothreitol (DIT). I
mmol/LEDTA, 1 X Denhardt'ssolution(0.04%eachpolyvinyl
pyrrolidone. bovine serum alhumine. and
Ficoll [Pharmncia. Uppsala. Sweden]). 1 0 % dextran sulphate. 0.1 mg herring sperm DNA/
mL and 0.5 mg tRNAlmL. Hybridization W;IS performed overnight
at 50°C in the same solution as was used for prehyhridization with
laheled prohes at aconcentrationof 2 X IO' cpm/pL. The slides
were washed in 4 X SSC ( I X SSC: 0. 15 mol/L NaCI. 0.015 moll
L Na citrate. pH 7.0). 10 mmol/L DTT and in 2 X SSC. I O mmoll
L DTT for 15 minutes at room temperature. I X SSC. I O mmollL
D I T for 30 minutes at 50°C and in 0 . I X SSC for 2 hours at 60°C.
then incuhated in RNAseA (10 pg/mL) for 30 minutes at 37°C in
0.5 mol/L NaCI. 10 mmolR. TRIS, pH 7.0. I mmollL EDTA. After
the tinul dehydration the slideswereimmersed
in Kodak NTB-2
photographic emulsion (Eastman-Kodak) and exposed at 4°C for 10
days. After developing, the cells were stained with Giemsa.
Corrrplc~r~lolfrtr~
D N A .s~rt/hc~.si.s
crrrcl / ~ o l ~ r r r ~ ~ clroirr
r ~ r s c rwc/iorr
(PCR) cr/rlplificcrrion. One to threemicrograms of total RNA extracted from BM cells was used for cDNA synthesis performed using
the First Strand cDNA synthesis Kit (Pharmacia. Uppsala. Sweden).
The reaction was primed with 200 ng of oligo-dT primer or with
h0 pmol o f PAEP-specilic primer. A negative control w a s included
in the first-strand synthcsis that contains the above reagents. but
noRNA.Cross-contanlination of RNA W:IS controlled hy reverse
transcription reaction that contains RNA and a11 reagents except
reversc transcriptase enzyme. Secretory cndometrium and TPA-induced K562 cells were used as positive controls. Amplilication of
the PAEPcDNA
was performed using the following primers:
S'CCAACAACATCTCCCTCATG.1' ( n t 165- 194.exon 2 ) fonvard
primer and S'GTGCAGGTGTGCAGCAGGAA.1' (nt 720-711.3'
untranslated region) reverse primer. Thirty cycles of PCR were performed on a Techne PHC-2 (Techne
Ltd. Cambridge. UK) thermocycler with thc following conditions: 95°C. 30 seconds: 55°C. 1 minute
30 seconds: 72°C. 2 minutes. To control the efficiency of reverse
transcription reaction. a control amplilication of the ubiquitous protein. cyclophilin. was pcrfornmetl. After amplification. the samples
were scpnratcd on a 1.5% agarose gel. stainedwith ethidium bromide
and photographed under ultraviolet light. Southern transfer and hyhridizntion with PPI4 cDNA"' probe were performed to confirm that
PAEP cDNA w a s specifically amplified.
RESULTS
Complementary DNA from BM cells was initially subjected t o reverse transcription (RT)-PCR using PAEP-specific primers. A major band of appropriate size (S67bp) w a s
visualized in ethidium bromide staining corresponding to
the size ofthepositivecontrol(Fig
I ). Specificity of the
amplified products was established by PAEP/PP14
cDNA
probe hybridization under stringent conditions (Fig I ).
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
KAMARAINEN ET AL
470
A
I
poietic cells. we used human leukemia cell lines with an in
vitrodiffcrcntiationpotential.Beforeinduction.
the K562
cells did not contain any matwc 0.9 kb PAEP/PP14 mRNA"'
detectable by Northernblotting.whereasa
faint band was
seen at 6 kb. Treatment of K562 cells with TPA for two
days induced dramatic accumulation of mature 0.9-kb PAEP
mRNA(Fig4).
Ahybridizationsignal
at 6kbwasalso
present in the TPA-treated K562 cells. The higher molecular
band may represent unprocessed PAEP/PP14 mRNA.
When cytocentrifuged smears of K562 cells were stained
by indirect immunofluorescence with anti-PAEP MoAbs. intense cytoplasmic reactivity was seen in the cells treated with
TPA for 3 days. Like in the BM normoblasts. the strongest
immunostaining in TPA-treated K562 cells was seen in the
Golgi area (Fig SA).Uninduced K562 cells gave no specific
fluorescence above background reactivity (Fig SB). whereas
cells induced with RA, or with sodium butyrate for 3 days.
displayed ;I wcak-positive staining (results not shown). Immunostainings of the megakaryoblastic cell lines Dami and
HPRC, the promonocytic cell line. U9.77. and the promyelocytic line HL-60 werenegativebeforeandafter
induced
differentiation (results not shown).
Immunoblot analysis with polyclonal rahbit anti-PAEP
B
.
Fig 4. Northern blot analysis of PAEP gene expression in K562
cell line. Poly A' RNA was extracted from (A) untreated and
(B1 TPAtreated cells. The positions of PAEP mRNA markers are indicated. A
rat GAPDH probe was used to ascertain that comparable amounts
of RNA had been loaded ineach lane.
In situ hybridization was performed to show PAEP genc
expression in BM. Strong hybridization signals wereseen
in a small population of
cells. Control hybridizations with
sense and nonsense probes were negative (Fig 2 ) .
After the detection of PAEP mRNAin normal BM, immunocytochemistry was applied to identify the cells containing
PAEP protein. When the cytocentrifuged smears of normal
BM werestained with anti-PAEPMoAbs by theindirect
peroxidase antiperoxidase (PAP) method, PAEP immunoreactivitywasfound in cells of theerythroidlineage.Early
maturation stages of the normoblast series displayed cytoplasmic andmembranestaining with the highestintensity
seen in the Golgi complex (Fig 3A and B). Double immunofluorescence with anti-PAEP MoAbs and rabbit antiglycophorin A showed that the samenucleated erythroid precursor
cells expressed both antigens (Fig 3D through F). Mature
RBCs and cells of the myeloid and lymphoid
lineages did
not stain with anti-PAEP MoAbs, whereas weak reactivity
was seen in some megakaryocytes. The antigen was not
detectable in platelets as studied by Western immunoblotting
(results not shown).
Immunoprecipitation with rabbit anti-PAEPantibodies
from detergent lysates of whole BM cells. followed by Westem immunoblot analysis. gave a band (Fig 3C) corresponding to the size of PAEPlPP14.'
To further investigate the expression of PAEP in hemato-
Fig 5. Staining ofPAEP by immunofluorescence in K562 cells
treated with TPA for 3 days. (A) Uninduced K562 cells are mainly
PAEP negative, but some spontaneously differentiated cells show
PAEP reactivity. (B) After 3 days' treatment with TPA, the cells are
consistently positive.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
47 1
PAEP IN HUMAN ERYTHROIDPRECURSORS
Fig 6. Western immunoblots using rabbit anti-PAEP antibodies of
lysates from (A) untreated K562 cells and from cells treated for 3
days with (B) RA, (C) sodium butyrate, and (D)TPA. The position of
PAEP is indicated by the arrow.
antibodies of lysates from TPA-treated K562 cells showed
a distinct bandthat comigrated with PAEP. Cells treated
withRA or sodium butyrate only gave barely detectable
bands (Fig 6).
K562 cells, either untreated or treatedwith TPA for 3
days, were metabolically labeled with "S-methionine for 2
hoursandlysed.
Immunoprecipitates obtained with antiPAEP MoAbs were separated on SDS-PAGE and analyzed
by autoradiography. A distinct band at 28 kD was seen in the
lanes containing precipitates from TPA-treated K562 cells,
whereas no specific radiolabeled bandwas obtained from
the lysates of control cells (Fig 7). Precipitation of the PAEP
bandwas largely abolished by the addition of excess of
purified decidual PAEPPP14"' to the lysate.
Immunofluorometric measurements showed that
the
m10-~
B
C
-*
'4--
21.5
Fig 7. Immunoprecipitation of PAEP protein from K562 cells. The
lysates were prepared from cells labeled for 2 hours with %-methionine and precipitated using anti-PAEP MoAbs. A shows the pattern
obtained from uninduced cells. B shows the pattern obtained from
TPA-induced K562cells. C shows the pattern obtained with antiPAEP MoAbs from TPA-induced K562 cells in the presence of 10 p g
unlabeled PAEP/PP14 protein.
0
24
48
72
96
120
time (hours)
Fig 8. Accumulation ofPAEP in the medium of TPA-treated (0)
and untreated (0)cultures of K562 cells.The bars indicate SE ofmean
values from quadruplicate cultures.
PAEP protein accumulates in culture medium of K562 cells
grown in the presence of TPA. PAEP became detectable
after 48 hours and its concentration increasedup to 120
hours of culture (Fig 8). No PAEP was found in the culture
media of untreated K562 cells (<3.1 pg/L).
DISCUSSION
There is a growing list of molecules expressed in both
the hematopoietic system and the reproductive tract. This
includes prolactin,".23 polypeptide growth factors,*' and tyrosine kinase receptors of the PDGFICSF-I receptor familY.25.'"
Results of the present study show that PAEP, previously
believedtobe
specific for thehuman reproductive tract,
is present in hematopoietic cells. Our immunocytochemical
findings indicate that PAEP is expressed in theerythroid
lineage. Positive staining was seen exclusively in the cells
representing different stages of maturation of the normoblastic series. Attempts to show the presence of PAEP in mature
RBCs gave negative results. Because of methodologic limitations of immunocytochemistry and the lowfrequency of
early erythroid precursors in normal BM, it remains to be
clarified whether PAEP is expressed already in proerythroblastic cells. This question is now being addressed.
In their preliminary work, Morrow andhis coworkers have
reported the occurrence of cDNA corresponding to PAEP/
PP14 in a cDNA library in the erythroleukemia cell line
K562." The present study confirms and extends this observation and lends support to the view thatthe initiation of PAEP
expression may occur at later stages of erythropoiesis. PAEP
cannot be detected in normal K562 cells, whereas treatment
of the cells for 2 to 3 days with compounds known to induce
in vitro differentiation brings about expression of the PAEP
message and the protein.
PAEP was found to accumulate in the culture supernatant
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
472
of differentiating K562 cells. The question of whether this
is because of release from disintegrating cells or results from
active secretion is nowunder investigation. The serum
PAEPPPl4 concentration is higher inwomen
than in
men.’,’’ Although the endometrium and the seminal vesicles
are believed to be the main sources, it cannot be excluded
that erythropoietic cells may contribute to the circulating
PAEP pool.
Details of the regulation of PAEP expression in erythroid
cells remains to be elucidated. Interestingly genes for human
PAEPPP14 and of the A B 0 blood group antigens are
mapped to the same chromosomal band 9q34.29 Karhi and
his coworkers3’ reported that during erythropoiesis, the expression of blood group A antigen occurs at the stage of
basophilic normoblast differentiation. The present findings
suggest that the expression of PAEP may coincide with that
of the major blood group antigens. However, whereas the
structures carrying the A B 0 blood group substance are abundant in mature RBCs, PAEP is not.
In view of the reported immunosuppressive functions of
PAEP, it is of interest to note that K562 cells, which are the
standard in vitro target for human NK cells, become virtually
resistant to NK cell-mediated lysis during induced differentia t i ~ n .In
~ ’that respect, the finding that PAEPPP14 inhibits
NK cell activity” on K562 cells is of great interest. It remains
to be studied whether the induced expression of PAEP contributes to the acquisition of NK resistance.
REFERENCES
I . Julkunen M, Koistinen R, Sjoberg J, Rutanen E-M, Wahlstrom
T, Seppala M: Secretory endometrium synthesizes placental protein
14. Endocrinology 118:1782, 1986
2. Julkunen M: Human decidua synthesizes placental protein 14
(PP14) in vitro. Acta Endocrinol 112:271, 1986
3. Julkunen M, Koistinen R, Suikkari A-M, Seppala M, Janne
OA: Identification by hybridization histochemistry of human endometrial cells expressing mRNAs encoding a uterine P-lactoglobulin
homologue and insulin-like growth factor-binding protein-]. Mol
Endocrinol 5:700, 1990
4. KiimWinen M, Leivo I, Julkunen M, Seppala M: Localization
of progesterone-associated endometrial protein mRNA by in situ
hybridization in human pregnancy decidua, endometriosis and borderline endometrioid adenoma. J Mol Endocrinol 10:71, 1993
5. Julkunen M, Wahlstrom T, Seppala M, Koistinen R, Koskimies
A, Stenman U-H, Bohn H Detection and localization of placental
protein 14-like protein in human seminal plasma and in the male
genital tract. Arch Androl 12:59, 1985
6. Bohn H, Kraus W, Winckler W: New soluble placental tissue
proteins: Their isolation, characterization, localization and quantification. Immunology of human placental proteins. Placenta 4:67,
1982
7. Joshi SG, Smith RA, Stokes DK: A progestagen-dependent
endometrial protein in human amniotic fluid. J Reprod and Fertil
60:317, 1980
8. Bell SC, Bohn H: Immunochemical and biochemical relationship between human pregnancy-associated secreted endometrial a,and a2-globulins ( a I- and a2-PEG)and the soluble placental proteins
12 and 14 (PP12 and PP14). Placenta 7:283, 1986
9. KamWinen M, Julkunen M, Seppala M: HinjI polymorphism
in the human progesterone-associated endometrial protein (PAEP)
gene. Nucl Acid Res 19:5092, 1991
10. Julkunen M, Seppala M, Janne 0: Complete amino acid se-
KAMARAINEN ET AL
quence of human placental protein 14: a progesterone-regulated uterine protein homologous to P-lactoglobulins. Proc NatlAcadSci
USA 85:8845, 1988
1 I . Papiz M, Sawyer L, Elipoulos E, North JA, Findlay J, Sirvaprasadarao R, Jones T, Newcomer M, Kraulis P: The structure of
P-lactoglobulin and its similarity to plasma retinol binding protein.
Nature 324:383, 1986
12. Seppala M, Riitinen L, Julkunen M, Koistinen R, Wahlstrom
T, Iino K, Alfthan H, Stenman U-H, Huhtala M-L: Structural studies,
localization in tissue and clinical aspects of human endometrial proteins. J Reprod Fertil Ster 36:127, 1988
13. Bolton AE, Clough K J , Stoker RJ, Pockley AG, Mowles EA,
Westwood OMR, Chapman MG: Identification of placental protein
14 as an immunosuppressive factor in human reproduction. Lancet
I:593, 1987
14. Pockley AG, Mowles EA, Stoker RI, Westwood OMR, Chapman MG, Bolton AE: Suppression of in v i m lymphocyte reactivity
to phytohemagglutinin by placental protein 14. J Reprod Immunol
13:31, 1988
15. Okamoto N, UchidaA, Takakura K,Kriya Y, Kanzaki H,
Riittinen L, Koistinen R, Seppala M, Mori T: Suppression by human
placental protein 14 of natural killer cell activity. Am J Reprod
lmmunol 26:137,1991
16. Anderson LC, Jokinen M, Gahmberg CG: Induction of erythroid differentiation in the human leukaemia cell line K562. Nature
278:364, 1979
17. Sutherland JA, Turner AR, MannoniP,McGannLE,
Turc
J-M: Differentiation of K562 leukemia cells along erythroid, macrophage, and megakaryocyte lineages. J Biol Response Mod 5:250,
1986
18. Riittinen L, Stenman U-H, Alfthan H, Suikkari A-M, Bohn H,
Seppala M: Time resolved immunofluorometric assay for placental
protein 14. Clin Chim Acta 183:115, 1989
19. Riittinen L, Nmanen 0, Virtanen I, Seppala M: Monoclonal
antibodies against endometrial protein PP14 and their use for purification and radioimmunoassay of PP14. J Immunol Methods 136:85,
1991
20. Riittinen L, Julkunen M, Sepptilij M, Koistinen R, Huhtala
M-L: Purification and characterization of endometrial protein PP14
from mid-trimester amniotic fluid. Clin Chim Acta 184: 19, 1989
2 I . Willebrand von E, Zola H, Hayry P: Thrombocyte aggregates
in renal allografts. Analysis with fine-needle aspiration biopsy and
monoclonal anti-thrombocyte antibodies. Transplantation 39:258,
1985
22. Pellegrini 1, Lebrun J-J, Ali S , Kelly PA: Expression of prolactin and its receptor in human lymphoid cells. Mol Endocrinol
6: 1023, 1992
23. Sabharwal P, Glaser R, Lafuse W, Varma S, Liu Q , Arkins
S, Kooijman R, Kutz L, Kelley KW, Malarkey WB:Prolactin synthesized and secreted by human peripheral blood mononuclear cells:
An autocrine growth factor for lymphoproliferation. Proc Natl Acad
Sci USA 89:7713, 1992
24. Pampfer S, Tabibzadeh S, Chuan FC, Pollard JW: Expression
of colony-stimulating factor-l (CSF-l) messenger RNA in human
endometrial glands during the menstrual cycle: Molecular cloning
of a novel transcript that predicts a cell surface form of CSF-I . Mol
Endocrinol 5:1931, 1991
25. Aprelikova 0, Pajusola K, Partanen J, Armstrong E, Alitalo
R, Bailey SK, McMahon J, Wasmuth J, Huebner K, Alitalo K: FLT4,
a novel class 111 receptor tyrosine kinase in chromosome 5q33-qter.
Cancer Res 52:746, 1992
26.Kaum SW, Aukerman SL, Eierman D, Turner T: Colonystimulating factor-] and c-fms expression in human endometrial
tissues and placenta during the menstrual cycle and early pregnancy.
J Clin Endocrinol Metab 73:746, 1991
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
PAEP IN HUMAN ERYTHROID PRECURSORS
27. Morrow DM, Getty RR, Riittinen L, Seppala M, Tykocinski
ML: Expression of the immunosuppressive molecule placental protein 14 (PP14) by the myelogenous leukemic cell line K562.Keystone Symposium on Molecular and Cellular Biology Tamarran,
Denver, CO, March 1, 1992
28. Julkunen M, Rutanen E-M, Koskimies A, Ranta T, Bohn H,
Seppdill M: Distribution of placental protein 14 in tissues and body
fluids during pregnancy. Br J Obstet and Gynaecol 92:1145, 1985
413
29. Van Gong N, Vaisse C, Gross M-S, Slim R, Milgrom E,
Bernheim A: The hwnan Placental Protein 14 (pp141 gene is localized On chromosome 9q34. Hum Genet 86:5159 1991
30. Karhi K, Adersson LC, Vuopio P, Gahmberg CC: Expression
of blood group A antigens in human bone marrow cells. Blood
57:147, 1981
31. Gidlund M, Om A, Pattengale PK, Jansson M, Wigzell H,
Nilsson K: Natural killer cells kill tumour cells at given stage of
differentiation. Nature 292:848, 1981