stimulus- response coupling may be relevant to unregulated cell growth in ..... a model to assess the role of neutrophil specific granules in the evolution.
Impaired stimulus-response coupling increased growth rate of HL6O cells Nicholas
Hadjokas,*
Clifford
Bayer,
Clinical Pharmacology and Gerontology 0Department of Pharmaceutical Sciences, and
Gerontology
Geriatric
Medicine,
and
Research College
Department
in association
Christopher
P. Nielsont
Unit, Department of Veterans Affairs of Pharmacy, Idaho State University, of Medicine,
with
University
Washington,
Idaho; of
Seattle
useful changes
an impairment in both the respiratory burst and secretion of 13-glucuronidase. In addition, regulation of the respiratory burst by CAMP agonists including isoproterenol, adenosine, and prostaglandin E2 was reduced in rapidly proliferating cells. Thus, multiple changes in stimulus-response coupling occur during cell culture in association with an increase in rate of cell replication. It may be important to recognize progressive impairments in cell function in studies using repetitive samples of HL6O cells from a continuously maintained cell
tent with the conclusion that repetitive passaging select for a subset of the cell population that rapidly, is least susceptible to differentiation, most pronounced impairments in cell function.
growth 157-160; Key
The coupling in
observed may be
neoplastic 1992.
Words:
respiratory
impairments relevant to
J.
disease.
burst
HL6O
in stimulusunregulated cell Biol.
Leukoc.
calcium
52:
cell culture
in
or effector experimental
leads
to cell
prolifera-
Because HL6O cells differentiate to a number cell types including neutrophils, macrophages, sinophils [1] and are inhibited by beta-adrenoceptor in a manner similar to human neutrophils, the
of myeloid and eoagonists cell line is
Selective coupling,
culture significantly the stability of cell
affect charac-
of cells may grows most and has the
HL6O Cells HL6O
cells
were
obtained
from
the
American
Type
Culture
Collection at passage 17. The cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (Sigma), 2 mM L-glutamine, 50 g/ml streptomycin, and 50 lU/mI penicillin. Fetal calf serum and water used to prepare the media were endotoxin tested ( < 0.25 ng/ml). The cells were passaged three times weekly to maintain cell density at approximately 1 x 106 cells/ml. Cells were differentiated to a neutrophil-like cell by 4 days exposure to 1.5% dimethyl sul-
Respiratory
regulation
cell
[2-7].
Methods
HL6O human promyelocytic leukemia cells were studied to determine whether cell characteristics are stable during a period of cell culture. Because HL6O cells are neoplastic, normal regulation of cell growth and differentiation is impaired in them. Studies were performed to determine whether (1) altered cell growth and (2) associated impairments in cell function and regulation are static or progres-
tion and the specific change is equally transferred to all subsequent cells, then impairments in cell growth and function may be constant. In contrast, if abnormalities in growth, differentiation, and function are variable between cells, then the most rapidly proliferating cells that transfer the characteristic of rapid proliferation to daughter cells would be expected to dominate progressively a cell culture population. A similar phenomenon could occur with neoplastic cell populations in vivo.
during Therefore,
investigations stimulus-response
teristics is relevant both to the mechanisms of neoplastic transformation and to the practical utilization of cultured cells. This report characterizes changes in the HL6O cell respiratory burst, secretion, and cAMP regulation during a 45-week period of cell culture. Our results are most consis-
foxide
with time. change in cell
range of responses,
activation results.
INTRODUCTION
sively altered If a specific
a broad receptor
Center, Boise, and tDivision
Abstract: Impairments in the respiratory burst and stimulus-response coupling were studied with respect to the increased rate of cell replication that occurred in HL6O cells during repetitive passages in cell culture. During a 45-week period of culture, HL6O cells developed a progressive increase in rate of replication. Concomitantly, undifferentiated cells developed an impairment in ATPinduced calcium mobilization. The percentage of cells that could be differentiated with dimethyl sulfoxide progressively diminished. Differentiated cells developed
population. response
for
of
Medical Pocatello;
HL6O
(DMSO).
cells
gation at resuspended cium detect
Burst were 300g in
and 5 mM oxygen
removed
from
for 10 mm phosphate-buffered glucose. metabolite
culture and
Lucigenin generation
media
4
x saline
with
centrifu-
10 cells/ml with 1 mM
(10 tM) and
was the
included cells
were calto were
stimulated with 1 M N-formylmethionyl-leucyl-phenylalanine ( fMLP). Lucigenin-dependent luminescence was then monitored for 20 mm at 37#{176}Cin a Packard Picolight luminometer. To evaluate the respiratory burst in individual cells, the HL6O cells were incubated for 25 mm with nitroblue tetrazohum (NBT) after stimulation with 10 nM phorbol myristate acetate (PMA). Reduction of NBT to a dark blue color was utilized to indicate activation of the respiratory burst.
Abbreviations: DMSO, dimethyl sulfoxide; IMLP, N-formylmethionylleucyl-phenylalanine; NBT, nitroblue tetrazolium; PMA, phorbol myristate acetate. Reprint requests: Christopher Nielson, Research Service (151), VAMC, 500 West Fort St., Boise, ID 83702. Received November 18, 1991; accepted February 11, 1992.
Journal
of Leukocyte
Biology
Volume
52,
August
1992
157
3-GIucuronidase Secretion
Secretion
was
assayed
with
a modification
of the
method
of
Gallin et al. [8J, which allowed measurement using microtiter plates. HL6O cells at a concentration of 5 x 106 cells/mi were stimulated with 1 M fMLP in HEPES buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KC1, 1 mM CaCi2, 1 mM MgCl2, 5.6 mM glucose) for 10 mm at 37#{176}C and then immediately cooled in an ice bath. After centrifugation at 300g for 10 mm, f3-glucuronidase activity was determined in the supernatant. Supernatant (20 l) was added to 90 jl phenolphthalein glucuronide substrate (1 ml 10 mM phenolphthalein glucuronic acid in 17 ml 33 mM acetate buffer, pH 4.5) and incubated 100 /Li 400 mM glycine buffer NaCI in 200 ml H2O, pH 10.5), quantitated secretion glucuronidase an identical
overnight. (3.26 gm /3-glucuronidase
After addition glycine, 2.53 activity
by was
measurement of absorbance at 550 standardized as the percentage released by cell lysis with 10% Triton unstimulated cell sample.
Intracellular
(1 for
x
were Fura-2
nm. of X-100
incubated with 3 iM was removed, and the
was obtained other materials St. Louis, MO.
from Molecular were obtained
from
Probes, Sigma
Eugene, Chemi-
40 GROWTH
RATE
P E R
c E
25
20 PASS Fig.
1. Growth
interval.
158
27
Growth of
rate
of
27
cells
from
each
passage
samples
PASS
Journal
by
passage
(P
