Impaired stimulus-response coupling in association with ... - CiteSeerX

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