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COMMUNICATION
Presence of Mason-Pfizer
Monkey Virus in Some Stocks
of the Human HBL-100
Mammary Epithelial Cell Line
Marjorie Robert-Guroff,
Theodore L. Stern, ErseIIS.
Richardson, Beppino C.
Giovanella, Frank H. Michaels*
The HBL-100 cell line, established
from primary cultures of milk epithelial
cells of a healthy woman (7,2), owes its
transformed phenotype to integrated
simian virus 40 (SV40) genetic information (3). The cell line lacked malignant
potential in early passages, but it acquired the ability to grow in soft agar
between passage 6 and passage 12; by
passage 100, it elicited tumors in nude
mice (3,4). HBL-100 cells have been
widely used as a model of malignant
progression and as a source of normal
human mammary epithelial cells (5-9).
Numerous investigations (10-17) in
the 1970s concerned a retroviral etiology of human breast cancer. A human
breast cancer virus was never isolated,
and interest in this possibility waned.
New, highly sensitive technology prompted
us to re-examine the retrovirus-breast
cancer hypothesis.
The product-enhanced reverse transcriptase assay (18) was used to screen
three established human breast cancer
cell lines, HTB20, HTB121, and
HTB126. All of these cell lines were
from the American Type Culture Collection (ATCC), Rockville, MD. HBL100 cells, designated HTB124 passage
27 (ATCC), were used as a control.
Only the HTB124 cell supernatant exhibited reverse transcriptase (RT) activity, which was enhanced following
cell treatment with iododeoxyuridine
372
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and dexamethasone (19) to induce retroviral expression (data not shown). A
subsequent standard RT assay (20)
showed that the HTB124 supernatant
enzyme was 13-fold more active with
dT|2_i8 • An a s template (1.3 x 105 cpm
dTMP [i.e., deoxythymidine monophosphate] incorporated/mL culture supernatant) compared with dT| 2 .i 8 • dAn (1.0 x
104 cpm/mL) and was 20-fold more active with Mg2+ cation (1.3 x 105
cpm/mL) compared with Mn2+ (6.5 x
103 cpm/mL). These properties are characteristic of RTs of human and nonhuman primate type D, murine type B,
and avian type C retroviruses. The
HTB124 supernatant was negative by a
laboratory antigen capture assay (Program Resources, Inc., National Cancer
Institute-Frederick Cancer Research and
Development Center, Frederick, MD)
for human immunodeficiency virus type
1 (HIV-1) and by the Coulter antigen
capture assay for simian immunodeficiency virus/HIV-2 p24 (data not
shown). Thus, the HTB124 cells were
not contaminated with either type of
HIV, which were also cultured in the laboratory.
The presence of RT activity in
HTB124 cells was intriguing and
recalled prior studies on RT in human
milk (16). A second vial of ATCC
HTB124 passage 27 cells was obtained,
and again the cells were RT positive
(9.1 x 104 cpm/mL with dT,2-i8 • An).
Following one passage, cytogenetic and
isoenzyme analyses carried out by Applied Genetics Laboratories, Inc. (Melbourne, FL), indicated that the cells
were of human origin (data not shown).
Transmission
electron
microscopy
showed typical type D retroviral particles in both aliquots of HTB124 cells
(Fig. 1). Infectivity of these particles
was demonstrated by transmission to Tand B-cell lines via HTB124 cell-free
supernatant or coculture with irradiated
HTB124 cells. Subsequent RT activity
with dT12.i8 • An in supernatants of the Tcell lines CEM and MOLT3 ranged from
2.4 x 105 to 5.2 x 105 cpm/mL; in the Bcell lines NC37 and Daudi, this activity
ranged from 7.0 x 104 cpm/mL to 1.8 x
105 cpm/mL. Viral particles were also observed by transmission electron microscopy in CEM cells cocultured with
HTB124 cells (Fig. 1).
Since HBL-100 cells are SV40 positive (3), the type D morphology suggested that contamination with a second
monkey virus may have occurred. The
retrovirus was examined for identity
with the following known type D retroviruses: squirrel monkey retrovirus
(SMRV) and Mason-Pfizer monkey
virus (MPMV). Type B murine mammary tumor virus (MMTV) was also investigated. Southern blot analysis
showed that the HTB 124 cells were not
infected with SMRV (Fig. 2, A). However, MPMV-specific products were
amplified by polymerase chain reaction
from HTB 124 and MPMV DNAs but
not from SMRV or MMTV DNAs (Fig.
2, B). Therefore, the retrovirus detected
in HTB 124 is MPMV or a highly related
retrovirus.
Early-, middle-, and late-passage
HBL-100 cells from viably frozen
stocks separately maintained by one of
us (B. C. Giovanella) were MPMV
negative (Fig. 2, C). However, all three
passages of HBL-100 cells were SV40
positive (Fig. 2, D), suggesting that the
MPMV contamination of HTB 124 cells
was independent of SV40 acquisition.
MPMV was originally isolated from a
spontaneous mammary carcinoma of a
rhesus monkey (21). While highly related simian type D retroviruses are
pathogenic and cause an AIDS-like disease in rhesus monkeys (22,23), there is
no evidence that MPMV is etiologically
associated with mammary carcinoma.
As shown here, the virus plays no role in
the malignant progression of HBL-100
cells. However, MPMV readily infects
human cells and has been reported in
humans (24-27), although its diseasecausing potential in humans is unknown.
Infection of cells with MPMV could
alter biological functions. Early-passage
HBL-100 cells possessed estrogen and
prolactin receptors and responded to
*Affiliations of authors: M. Robert-Guroff, T. L.
Stern, E. S. Richardson, F. H. Michaels,
Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, MD: B. C. Giovanella, The
Stehlin Foundation for Cancer Research. Houston,
TX.
Correspondence to: Marjorie Robert-Guroff.
Ph.D.. National Institutes of Health. Bldg. 37. Rm.
6A09, Bethesda. MD 20892-4255.
See "Notes" section following "References."
Journal of the National Cancer Institute. Vol. 88, No. 6, March 20, 1996
Fig. 1. Type D retroviral particles in
HTBI24 cells and in CEM cells following coculture with irradiated
HTB124 cells. Cells were fixed in
glutaraldehyde. postfixed in osmium
tetroxide, en bloc stained with uranyl
acetate, dehydrated in a series of
graded ethanols, and infiltrated and
embedded in Spurr's plastic resin.
Prior to examination, ultrathin sections on grids were post-stained with
lead citrate. The four pictures to the
left illustrate from top to bottom an
intracistemal A-type panicle (arrowhead), a budding virion (short
arrow), an immature type D particle
(double arrowhead), and a mature
type D panicle (long arrow) in
HTB124 cells (original magnification
X90 000). The top right picture is a
lower magnification (original magnification x22 500) of HTB 124 cells
in which three of the four type D
forms can be seen. The bottom right
picture shows a CEM cell to which
the retrovirus has been transmitted;
intracistemal A-type panicles are
marked with arrows, and extracellular particles are seen at the top
(original magnification X9000).
hormones with casein production (1,28).
HTB124 passage 26 cells, however,
lacked both receptors and were unresponsive to hormones (S). Whether
contaminating MPMV played a role in
down-regulating (i.e., decreasing) these
receptors requires further study.
HTB124 cells produce abundant retroviral particles, as judged by the ease
of their detection by transmission electron microscopy and their ready transmission. Users of HBL-100 cells should
be aware that some cell stocks are
MPMV producers.
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373
Fig. 2. Molecular analyses of
HTB 124 and HBL-100 cells. A)
A
B
Southern blot showing the presence
Hind III
Bam HI
of squirrel monkey retrovirus
i
II
o
1
(SMRV) type D provirus in SMRV>
j , CM
>
CM
c
CM
>
>
>
>
producer cells but not in HTB 124 or
t
DC m
o
oc m
t
£
5
S
Q_
other control cells. Genomic DNA
ho
I
5
c5o iK 05 2 CO X
|
was extracted from cells (29), includi
ing those producing known retro23.5-23.5
•
MPMV-env
•
viruses: human A204 rhabdomyosarcoma cells producing MasonMPMV-gag
—
•
Pfizer monkey virus (MPMV),
DPSO 114/74 (ATCC CCL 194)
squirrel monkey lung cells producing
o
-5.8
in
SMRV, and Mm5MT (ATCC CRL
Q. a. a.
o O
1637) mouse mammary gland cells
c o O 8
4.2o
producing murine mammary tumor
Zi
>
u m m m
-3.6
virus (MMTV). DNA (30 ug) was
X
I
I
co :
digested with 150 U of either BamHl
3.1 MPMV-env
or Wmdlll (Boehringer Mannheim
Corp., Indianapolis, IN). Following
electrophoresis, blotting, and prehyMPMV-gag
bridization, filters were hybridized
with the plasmid probe pSMRV (30)
D
provided by Dr. Steve Tronick, Nao
in
tional Cancer Institute. Hybridization
•g
a. Q.
o
was carried out overnight at 37 'C
>
CM
c
o
6
o
using 5 x 10 cpm/mL of probe in
rr a. m
o
m CO m
Hybrisol I (Oncor, Inc., GaithersSO
C
X
I
I
X
—
burg, MD). The filters were washed
1.0at 60 °C in 3x SSC (lx SSC = 0.15
i
* • . SV40 Large T
M NaCl and 0.015 M sodium citrate)
containing 0.1% sodium dodecyl sulfate (SDS) and autoradiographed. B) Polymerase chain reaction (PCR) detecGCTTCCCAATATGG-3') probe. Filters were washed three times for 10
tion of MPMV env and gag genes in MPMV producer cells and HTB 124 cells.
minutes at 50 °C in 3x SSC containing 0.1% SDS and autoradiographed. C)
PCR analysis was carried out using env and gag primers described by BohanPCR detection of MPMV env and gag genes in MPMV producer cells but not
non et al. (24): for gag, 5'-TCCCAGTGACTGAAACCGTGATGGGC-3' and
in early, middle, or late passages of HBL-100 cells (maintained by B. C.
5'-CATGGCCAGGCCTTGCTG-3'; for env, 5'-CTCAATGCTTCCCAACCCGiovanella)
or in control cells. D) PCR detection of the gene for the simian
AGTTTAGCC-3' and 5'-GGAGGCTGTAGAAACGTTATAATAG-3'. The
virus 40 (SV40) large T antigen in all passages of HBL-100 cells and in
PCR reaction mixtures containing 0.5 (ig of DNA and 0.5 |ig of each primer
HTB 124 cells, but not in control cells. PCR was carried out using similar cywere initially heated at 94 °C for 3 minutes, subjected to 30 cycles of
cling times and hybridization and washing conditions as used for the MPMV
amplification (1 minute at 94 °C, 1.25 minutes at 50 °C, and 1.5 minutes at 72
PCR. The upstream primer was SV1 (5'-CTGAAATGAGCCTTGGGACT-3/),
*C), and elongated a final 7 minutes at 72 'C. Amplification products were
and
the downstream primer was SV2 (5'-GAGTTGCTTGGCTACACTGT-3').
electrophoresed, blotted, and hybridized in Hybrisol II with an MPMVSV3 (5'-CACTCCACAATTCTGCTCTG-3') was the probe.
specific env (5'-CTGTGCTTACAGTCAGGAGA-3') or gag (5'-ACAGCT-
c
•
11
f
•• •
>
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1: 1
M1
•
•
8 8
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Notes
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Detection of an antigen related to MasonPresent address: F. H. Michaels, Institute of BioPfizer virus in malignant human breast
technology and Advanced Molecular Medicine,
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Thomas Jefferson University, Philadelphia, PA.
Chopra H, Ebert P, Woodside N, Kvedar J,
We thank Dr. Bemhard Kramarsky for the transAlbert S, Brennan M. Electron microscopic
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zuca for editorial assistance.
Morozov VA, Saal F, Gessain A, Terrinha A,
Manuscript received September 19, 1995; revised
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Journal of the National Cancer Institute, Vol. 88, No. 6, March 20, 1996