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thesis entitled
Alkaline Phosphatase and Retinoic Acid-Induced
Differentiation of HL-6O Cells

presented by

Deborah Ann Swartz

has been accepted towards fulfillment
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ALKALINE PHOSPHATASE AND RETINOIC ACID-INDUCED
DIFFERENTIATION OF HL-6O CELLS

BY

Deborah Ann Swartz

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1988

 

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Zr
07'

ABSTRACT
ALKALINE PHOSPHATASE AND RETINOIC ACID-INDUCED
DIFFERERENTIATION OF HL-6O CELLS
BY

Deborah Ann Swartz

Retinoic acid (RA) induces terminal
differentiation of HL-60 cells, however; the mechanism
is unknown. RA also induces alkaline phosphatase (AP)
activity in several normal and tumor cells. Since
increased AP activity is associated with highly
differentiated cells, the objective of this study was
to determine if RA-induced differentiation of HL-60
cells is associated with induction of AP activity.
HL-60 cells were exposed to RA (luM) for 0,1,12,24,36
and 72 h and DMSO (180mM), 1,25-(OH)2D3 (lOOnM) and
dexamethasone (lOOnM), for 0,24 and 72 h. Cells were
harvested at the indicated times, AP activity
determined, and differentiation assessed. by' NBT-dye
reduction and positive reactivity for chloroacetate
esterase staining. AP activity was not detectable in
HL-60 cells prior to and following treatment with any
of the above compounds. The results suggest that HL-60
cells lack AP activity ; this may contribute to

expression of their malignant phenotype. It appears

 

 

 

that RA-induced differentiation of HL-60 cells does not

involve alkaline phosphatase.

 

To my parents, Milton and Lorraine Swartz, for all

their love and support throughout my graduate program.

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to Dr.
Maija H. Zile, my major professor, for her support,
advice and criticism throughout my graduate program and
my sincere graditude to the members of my guidance
committee: Dr. M. Bennink, Dr. W. Chenoweth and Dr. C.
Welsch. I would also like to thank: Dr. P. Bank, Dr.
M. Cullum, H. Dersch, I. Roltsch and K. Salyers for all
their support, encouragement and friendship during my
graduate program. I couldn't have made it without

them.

 

 

TABLE OF CONTENTS

Page

List of Tables .............. . .......................... viii
List of Figures .......... . .......... . .................. ix
List of Abbreviations .......... ..... ................... X
I. INTRODUCTION ............... ..... ................. 1

II. REVIEW OF FUNCTION OF VITAMIN A AND RETINOIDS.... 4

 

A. Physiological Functions of Vitamin A ......... 4
B. Effects on Non-neoplastic Cells .............. 6
l. Epithelial cells ......................... 6
a. Keratinocytes ............ . ........... 6
b. Mammary gland ........................ 8
c. Testicular cells ..................... 8
2. Mesenchymal cells ........................ 9
a. Adipocytes ........................... 9
b. Chondrocytes ......................... 10
c. Fibroblasts .......................... 12
C. Effects on Neoplastic Cells .................. 13
1. Embryonal carcinoma cells ................ 13
2. Murine melanoma cells .................... 15
3. Leukemia cells- HL-60 Promyelocytes ...... 16
D. Summary ............. . ........................ 19
III. REVIEW OF FUNCTION OF ALKALINE PHOSPHATASE ....... 22
A. Physical and Chemical Properties of Alkaline
Phosphatase ................................. 22
B. Phosphoprotein Phosphatases and Alkaline
Phosphatase ................................. 23
C. Protein Phosphorylation/Dephophorylation in
Regulation of Growth and Differentiation ..... 24
D. Regulation of Alkaline Phosphatase by
Retinoids .................................... 28
E. Summary ......................... . ............ 33
IV. MATERIALS AND METHODS ............................ 34
A. Material Sources ............................. 34
B. Cells and Culture ............................ 35
C. Assessment of Differentiation .......... . ..... 36
D. Isolation of Human Granulocytes .............. 37
E. Preparation of Intestinal Mucosa ............. 38
F. Alkaline Phosphatase Assay ...... . ..... . ...... 39
vi

 

Table of Contents (continued)

VI.

VII.

VIII.

Page
G. Protein Assay ...... i ................. ' ......... 40
RESULTS.. ................. . ...................... 41

A. Treatment of HL-60 Cells with Retinoic Acid.. 41
B. Control Experiments with Cells Known to have

Alkaline Phosphatase Activity........ ........ 41
C. Effect of Known Inducers on Alkaline

Phosphatase Activity in HL-60 Cells ......... 48
DISCUSSION.. ...... . ..................... . ......... 57
CONCLUSIONS ...................................... 62
REFERENCES ....................................... 63

vii

 

 

LIST OF TABLES

Table Page

I. Effects of Retinoic Acid on Growth Rate and
Differentiation of Several Cell Lines........ 20

II. Alkaline Phosphatase Activity in HL-60 Cells
Following Treatment with Inducers ............ 49

viii

 

 

Figure

1.

LIST OF FIGURES

Page
Effect of all-trans-Retinoic Acid on the
Induction of Differentiation of HL-60 Cells
as a Function of Time ........................... 43
Napthol AS-D Chloroacetate Esterase Activity
in Retinoic Acid Treated HL-60 cells ............ 45
Alkaline Phosphatase Activity in a Variety
of Cell Types ....... ....................... ..... 47
Effect of 1,25(OH)2Vitamin D3 on the Induction
of Differentiation of HL-60 Cells as a Function
Of Timeooooooo0.000000000000000 000000000 o ooooooo 51
Effect of Retinoic Acid and Dexamethasone on
the Induction of Differentiation of HL-6O
Cells as a Function of Time.... ....... .... ...... 54

Effect of Retinoic Acid and Dimethyl Sulfoxide
on the Induction of Differentiation of HL-6O
Cells as a Function of Time....... .............. 56

ix

Iv-

.. - nun-If""'

 

 

AP
pBTM
CRABP

CRBP
CS
DMF
DMMEM

DMSO
EC
FBS
GAG
HPR
MSH
NBT

OPL
PBS
PMA
P-Ser
P-Thr
P-Tyr

TCA
TPA

LIST OF ABBREVIATIONS

alkaline phosphatase
l-p-bromotetramisole

cellular retinoic acid-binding
protein

cellular retinol-binding protein
chondroitin sulfate
dimethylformamide

Dulbecco's modified minimal
essential medium
dimethylsulfoxide

embryonal carcinoma

fetal bovine serum
glycosaminoglycan
N(4-hydroxyphenyl)-retinamide
d-melanocyte stimulating hormone
nitroblue tetrazolium
superoxide anion

outer plexiform layer

phosphate buffered saline
phorbol 12-myristate-13-acetate
phospho-serine
phospho-threonine
phospho-tyrosine

retinoic acid

trichloroooacetic acid
lZ-O-tetradecanoyl phorbol-13-
acetate

INTRODUCTION

Vitamin A is an essential micronutrient and 'is
involved in normal proliferation and differentiation of
cells of ectodermal, mesodermal and endodermal origin
(1,2). The term "vitamin A" is used generically to
include all natural compounds which exhibit the
biological activity of all-trans retinol, such as
retinaldehyde and retinoic acid. Retinoic acid is a
potent. differentiating agent and is known to alter
proliferation and differentiation of many cell types,
including normal and abnormal haematopoietic cells (3).
Although the expression of various proteins [has been
linked to vitamin A-induced differentiation (4), the
mechanism of action of vitamin A in differentiation is
still unknown.

Increased levels of alkaline phosphatase (EC
3.1.3.1) (AP) are associated with highly differentiated
cells such as Chondrocytes, the absorptive epithelium
of intestine and renal brush border epithelial cells
(5). Chondrocyte membrane AP selectively hydrolyzes

tyrosine phosphorylated phosphoproteins (5). The

phosphorylation of these proteins appears to be part of
the mechanism for signalling cells to undergo division,
hallowing the cells to differentiate (5).

Recent studies have shown that retinoic acid (RA)
induces .AP activity in both. normal and. tumor cell
systems, including normal rat prostatic and rat
prostatic adenocarcinoma cells (6), normal rat
urothelial tissue (7), and normal human neutrophilic
granulocytes (8). other agents, such as 1,25-
dihydroxyvitamin D3 and the synthetic glucocorticoid
hormone, dexamethasone, also induce AP activity in a
variety of different cell types (9,10). However, the
mechanism(s) involved are not well understood.

The human cell line HL-6O was isolated from the
blood of a patient with acute promyelocytic leukemia
and was the first human cell line with distinct myeloid
features to be developed (11). Retinoic acid, as well
as 1,25-dihydroxyvitamin D3 and dimethyl sulfoxide,
induce terminal differentiation of HL-60 cells into
cells with the morphological features of mature
monocytes (12) or granulocytes (13). Again, the
mechanism(s) involved remain largely unknown.

The jpurpose of ‘this study' was to test the
hypothesis that retinoic acid establishes
differentiation of the human promyelocytic leukemia

cell line, HL-60, through the induction of AP activity.

 

Alkaline phosphatase may act in. this cell line by
dephosphorylating' certain regulatory' growth factors,
thus, allowing the cells to differentiate.

The specific objectives of the study were to:

1. Determine if retinoic acid induced differentiation
of HL-60 cells is associated with the induction
of AP activity.

2. Determine if retinoic acid induced AP activity in
HL-60 cells, is correlated with an increase
in differentiation of the cells towards a more
normal phenotype.

3. Determine if AP activity can be induced in HL-60
cells by other differentiation agents and/or by
known inducers of AP activity.

The physiological function of alkaline phosphatase is

not known. If AP were involved in the regulation of

growth factors, this would explain the ubiquitous

nature of this enzyme.

 

REVIEW OF FUNCTION OF VITAMIN A AND RETINOIDS

A. Physiological Functions of Vitamin A

Vitamin A is an essential micronutrient found
primarily in plant foods in the form of the provitamin
B-carotene or in animal tissues as fat soluble retinyl
esters. The vitamin is required for normal
development, growth, reproductive capacity and vision.
With the exception of the visual cycle, very little is
known about the specific biological functions of
vitamin A on the molecular level. However, it has been
well established that retinoids, a family of molecules
comprising both natural and synthetic analogs of
vitamin A, are potent agents involved in cellular
differentiation and cellular proliferation (4).
Wolbadh and Hewe (14), in their classic paper
describing the effects of vitamin A deficiency in the
rat, reported distinct effects on both differentiation
and proliferation of epithelial cells. During vitamin
A deficiency , it was found that stem cells did not
differentiate normally into mature epithelial cells.
Instead, abnormal differentiation, characterized by

accumulation of keratin, was observed, along with

I '-
I‘b'v

 

excessive cellular proliferation in vitamin A deficient
epithelia. Later studies, both in vivo and in vitro,
confirmed these findings.

One of the most studied target tissues of vitamin
A has been the trachea. During vitamin A deficiency,
tracheal basal cell numbers increase, eventually
leading to squamous metaplasia (15). Upon retinoid
treatment, this condition is reversed (15), thus
demonstrating the importance of vitamin A in
maintaining the integrity of epithelial tissues.
Vitamin A not only affects the cellular proliferation
and differentiation of epithelial cells, but also that
of cells of mesenchymal origin. Mellanby (16) showed
that. retinoids affect. osteoblast. and osteoclast
activity, suggesting that vitamin A plays a role in
bone formation. Vitamin A is also important for the
development of the embryonic vascular system (17), as
well as for haematopoiesis (3).

Several in vitro studies involving many different
cultured cell lines, both neoplastic and non-
neoplastic, have shown that retinoids elicit many
diverse and often opposite effects on cells. For
example, in certain cell systems retinoids act to
inhibit cell growth, while in others they stimulate
cell growth. Retinoids can promote differentiation of

some cells and suppress it in others. These studies

 

have clearly shown that retinoids play a role in
regulating both cellular proliferation and

differentiation.
B. Effects on Non-neoplastic Cells

Retinoids have been shown to exert effects on
epithelial and mesenchymal cells in culture, similar to
those observed in vivo. However, it is not yet known
if retinoids act by the same mechanism in vivo and in

vitro.

1. Effects on Epithelial Cells
a. Keratinocytes

Recent studies involving primary cultures of
keratinocytes have shown that retinoids suppress the
formation of stratified squamous epithelia and
stimulate the formation of mucus secreting epithelia.
In particular, Fuchs and Green (18) found that vitamin
A regulates terminal differentiation of epidermal
keratinocytes. Basal cells divide and. move upward
synthesizing keratin and keratohyalin. They eventually
lose their nuclei, die and are shed from the uppermost
layer of the tissue. Fuchs and Green (18) showed that
the deepest cells synthesize keratins of molecular

weight 46-58 K dal. The upper layer or stratum corneum

1‘.- “EI‘.

 

forms keratins of 67 K dal. Green (19) showed that
human epidermal keratinocytes, when. cultured in the
presence of 10-20 % fetal bovine serum containing 4-8 x
10'8M retinol equivalents, undergo terminal
differentiation but they lack a stratum corneum and do
not synthesize the 67 K dal keratin. However, when the
cells were grown in medium containing 10% delipidized
serum, they underwent stratification and formed a
stratum corneum which is"“ characteristic of terminal
differentiation. Presence of the 67 K dal keratin was
detected after 2 weeks and levels were found to
increase 8.5% after four weeks. At the same time,
synthesis of the 40 K dal and 52 K dal keratins
decreased. Addition of 1.2 x 10-6M retinyl acetate to
the: delipidized. medium. reversed these effects. In
vitro translation of isolated keratin mRNA showed that
vitamin. A regulates the processes involved in
determining the amount of keratin-specific mRNA present
(19). The mRNA from cells grown in delipidized medium
directed translation of the 67 K dal keratin, while the
52 K dal keratin was translated from mRNA of cells
grown in the presence of retinyl acetate. These
results may be due to a direct effect by vitamin

A on transcription.

 

b. Mammary Gland

Mouse mammary glands can be induced to undergo
differentiation in organ culture by the addition of the
appropriate hormones to the medium (20). Extensive
end-bud development of the gland occurs when incubated
with insulin and prolactin, with some end buds
proliferating into alveolar structures. In the absence
of prolactin, such structures do not develope. The
addition of N(4-hydroxyphenyl)-retinamide (HPR) or RA
at a concentration of 1 uM inhibits prolactin-induced
differentiation of mouse mammary glands (21). In
contrast to this, retinyl acetate is ineffective at
1 uM and toxic at higher concentrations. These results
are consistent with published in vivo data. Welsch
et a1 (22) showed that HPR was an effective inhibitor
of mouse mammary gland tumorigenesis, however, retinyl
acetate was ineffective. Retinoids may modulate
differentiation and/or proliferation of mammary

epithelium in vitro.

c. Testicular Cells

Mather (23) isolated and cultured two
testicular cell lines of Leydig (TM3) and Sertoli (TM4)
origin. Upon treatment of these cells with RA or
retinol (10-7M), the growth rate of TM4 cells increased

significantly, while the growth of the TM3 cell line

was inhibited. The growth of a transformed variant of
the TM4 cell line was also inhibited with retinoid
(10‘8M) treatment. These examples illustrate the many
diverse responses to retinoid treatment not only in
different cell types but also in subclones of normal

and transformed cell lines.

2. Effects on Mesenchymal Cells
a. Adipocytes

Terminal differentiation of murine preadipocytes
into mature adipocytes is blocked by retinoids in a
dose dependent manner (24,25). Murray and Russel (24)
examined the role of RA in modulating the
differentiation of 3T3-L2 fibroblasts into adipocytes.
Differentiation of the preadipocytes was brought about
by treatment with l-methyl-B-isobutyl xanthine (0.5 mM)
plus dexamethasone (0.25 uM). In order to block
differentiation, RA (lo-llM to 10-5M) had to be
administered simultaneously with the triggering agents.
At 10'6M, RA inhibited the morphological and enzymatic
(fatty acid synthase activity) expression of the
adipose phenotype by more than 90%. Sato et al (25)
conducted a similar study using ST13 murine
preadipocytes induced to differentiate with insulin.
Treatment with RA or retinol at concentrations up to

10'5M had no effect on ST13 cell morphology, growth

 

10

rate or saturation density. However, accumulation of
triglycerides and increased cellular insulin binding
activity, both markers of preadipocyte differentiation,
were inhibited at retinoid concentrations as low as
10'8M. Upon removal of the retinoids cell
differentiation resumed. These studies suggest that
adipose tissue may be one of the target tissues for
vitamin A.
b. Chondrocytes

The differentiation of chondrogenic cells is
inhibited when cultures of cells derived from embryonic
cartilage forming cells are treated with RA (10'5M)
(26-28). Pennypacker et a1 (27) reported that mouse
embryo limb bud mesenchymal cells grown in culture
in the presence of 3 ug/ml RA inhibited the synthesis
of chondroitin sulfate (CS) proteoglycans, which are
markers of Chondrocyte differentiation. However, their
presence was detected in the untreated cultures. Under
similar experimental conditions, Kochhar et al (29)
reported a virtually complete suppression of CS
glycosaminoglycans (GAG) when treated with 1 ug/ml RA.
Seventy-two percent of the 3[H]-glycosamine-labeled
products, comprised of glycopeptide, hyaluronic ‘acid
and GAGs, were found in the culture medium of cells
continuously exposed to RA for 96 h. The amount of

labeled hyaluronic acid released into the medium by the

ll

RA-treated cells was almost 2 fold greater than that
released by the untreated cells. The authors noted
that this increase was not due to a greater than normal
synthesis of hyaluronate, but rather due to its
dislocation from the cell layer to the culture medium.
They suggested that this displacement may play a role
in the inhibition of Chondrocyte differentiation.
Trechsel et al (30) showed that dedifferentiation of
rabbit articular Chondrocytes induced by treatment with
retinol (10-6M), resulted in changes in collagen
synthesis from type II to type I collagen. These
results contrast with a more recent report by Benya and
Padilla (31). Rabbit articular Chondrocytes grown in
primary monolayer culture were treated with
0.03 to 3.0 ug/ml RA for up to 18 days. This resulted
in a loss of differentiated functions including: an
altered morphology, suppressed colony formation and
decreased proteoglycan (8 fold) and collagen
synthesis. Retinoic acid caused a decrease in type II
collagen synthesis, and a transient increase in
type III and type I trimer collagen synthesis; however,
type I collagen was not induced. Similar effects were
obtained with retinol. The authors suggest that the
discrepancy may be due to Trechsel's et al (30) use of
secondary cultures in which the Chondrocytes may have

already undergone subculture-dependent alterations in

12

their 'phenotype. The above studies suggest that
differentiation of chondrogenic cells is inhibited by
exposure to RA possibly due to changes in cellular GAG
biosynthesis.
c. Fibroblasts

Retinoic acid affects 3T3 and 3T6 murine
fibroblastic cell lines by decreasing growth rate,
altering cellular morphology, and increasing cell-to-
substratum adhesiveness (32). Retinoic acid treatment
of 3T6 cells leads to a reduction in saturation density
and a concentration-dependent increase in generation
time. Analysis of cell surface proteins indicated
levels of GAGs increased upon RA treatment. The
authors suggested that these increased leveLs of GAGs
may be involved in the enhanced adhesiveness, the
reduction in saturation density and the flattening of
the cells that were observed following RA treatment. A
RA binding protein was detected in the cytosol of both
the 3T3 and 3T6 cell lines, possibly acting as a
mediator for the action of retinoids.

Primary cultures of human fibroblasts also respond
to treatment with RA and retinol (10'8M to 10-5M) with
decreases in growth rate and saturation density (33).
However, cell morphology and cell-toasubstratum

adhesiveness remained unchanged after retinoid

13

treatment; no cytoplasmic retinoid binding protein

could be detected.

C. Effects on Neoplastic Cells

Retinoids have effects on the growth and

differentiation of a number of cultured neoplastic cell

lines. Neoplastic cells of epithelial (trachea,
bladder, breast carcinoma), mesenchymal (myeloid
leukemia, osteocarcinoma, chondrosarcoma) and
ectodermal (melanoma, embryonal carcinoma) origins

respond to retinoid treatment. Examples of retinoid
effects on neoplastic cells from various origins are

discussed below.

1. Embryonal Carcinoma Cells

Embryonal carcinoma (EC) cells, the stem cells of
teratocarcinoma, have the potential to differentiate
along pathways of each of the three basic germ layers
(34). Therefore, EC cell lines provide excellent in
vitro models for studying the processes by which
multipotential cells become committed.tx> a particular
pathway of differentiation. Strickland and Mahdavi
(35) found that RA can promote terminal differentiation
of murine F9 embryonal carcinoma cell line to 'non-

neoplastic endodenm. They were able to determine the

 

14

degree of differentiation using plasminogen activator
as a marker. It was found that optimal differentiation
occurs at 72 h and is RA concentration-dependent, with
detectable responses at concentrations of RA as low as
10'9M. F9 cells treated with RA will differentiate
into two cell types depending upon whether they are

grown as cell aggregates or as monolayer cultures.

Cell aggregates grown in the presence of RA
express °(-fetoprotein, a marker indicative of
visceral endoderm (36)- Monolayer cell cultures

exposed to RA followed by dibutryl cAMP, give rise to
differentiated cells characteristic of parietal
endoderm (37). Changes in morphology as well as in the
pattern of protein synthesis occur upon differentiation
of F9 cells with RA. Proteins characteristic of
parietal endoderm such as type IV collagen and laminin
are synthesized (37). Levels of cellular retinol-
binding protein and cellular retinoic acid-binding
protein are also elevated (38); however, the cells do
not appear to be dependent on retinoids to maintain
their differentiated state.

Changes in gene expression occur during the F9
differentiation process. However, most of these
changes take place after the actual induction of
differentiation (35). Campisi et a1 (39) showed that

myc. proto-oncogene (c-myc) mRNA greatly diminishes

 

15

after F9 cells are induced to terminally differentiate
with RA and cAMP. Dean et a1 (40) recently showed
that F9 cells treated with RA induced an early post-
transcriptional change in myc gene expression and that
this correlated with growth inhibition rather than
differentiation.

The effects of RA on the differentiation of F9
embryonal carcinoma cells can also be seen in vivo.
Sherman et al (41) injected mice with PCC4-aza 1R EC
cells or with the same cells treated with RA in vitro.
All the mice injected with the untreated cells
developed palpable tumors within 10-12 days. Six of 8
mice injected with RA-treated cells did not develope
palpable tumors even after 100 days. Therefore,
retinoids greatly reduce the malignancy of these cells

by inducing their differentiation.

2. Murine Melanoma Cells

Retinoic acid has been shown to reversibly inhibit
cell growth and stimulate melanin production in mouse
melanoma cells (42). Lotan and Lotan (42) treated S91
mouse melanoma clone C2 cells with RA at concentrations
ranging from 10'7 to 10'5M. Inhibition of growth,
which was detected only after 48 h of treatment, was
found to be dependent on the concentration of RA.

Ludwig et al (43) obtained similar results in B16-Fl

16

mouse melanoma cells. melanogenesis, which was
measured by tyrosine activity and melanin production,
was ~increased 3-4 fold by treatment with RA. In
contrast, exposure of a number of human melanoma cell
lines, e.g. UCT-Me 12, to RA inhibits melanogenesis
(44). Retinoic acid-induced melanogenesis was
distinguished from that induced by °(-melanocyte
stimulating hormone (MSH) (2 x 10'7M), in that RA
treatment did not alter the intracellular cAMP level,
whereas MSH induced a transient 4 fold increase in
cAMP concentration. Lotan and Lotan (42) also
investigated the effects of other retinoids such as
13-cis-retinoic acid and retinyl acetate on S91
melanoma cells. These retinoids also inhibited cell

growth and enhanced melanin synthesis.

3. Leukemia Cells - HL-60 Promyelocytes

Proliferation of haematopoietic cells is regulated

by a combination of growth inhibition and
differentiation ; however , the precise mechanisms
involved are not known ( 1 1) . In recent years the

development of leukemia cell lines has provided useful
models for studying cell proliferation and
differentiation. One cell line in particular, HL-60,
which was established from the peripheral blood of a

patient with acute promyelocytic leukemia (11),

 

17

provides a unique and useful model system due to its
inducible characteristics. HL-60 cells grow
continuously in suspension culture and consist
predominantly of promyelocytes. They can be induced to
differentiate into morphologicallymature granulocytes
by a variety of compounds including dimethylsulfoxide
(DMSO), dimethylformamide (DMF), hypoxanthine and RA
(45,13). These induced cells have 'many functional
characteristics of normal human peripheral
granulocytes, including phagocytosis, lysozomal enzyme
release, hexose monophosphate shunt activity, the
generation of superoxide anion (02') and the ability to
reduce nitroblue tetrazolium (NBT) (45-47). Other
compounds such as 1,25- dihydroxyvitamin D3
and 12-O-tetradecanoylphorbol-13-acetate (TPA) induce
HL-60 cells to differentiate into monocyte-like or
macrophage-like cells, respectively (48,49).

The finding that RA induces HL-60 cells to
differentiate (13) was important for subsequent
investigations since detectable responses could be seen
at physiological concentrations. Prior to this, the
only' known inducers ‘were, either' non-physiologic
compounds (DMSO) or physiological compounds
(hypoxanthine) that required concentrations much higher

than physiological.

 

18

Retinoic acid differentiates HL-60 cells in a
concentration-dependent and time-dependent manner (13).
The greatest degree of differentiation occurs when
HL-60 cells are incubated continually for 3 days at a
RA concentration of 10'6M (13). At this point the
cells no longer proliferate, but maintain their
differentiated state even after they are resuspended in
RA-free medium . Studies from our laboratory
demonstrated that an exposure to RA for only 30 min.
was sufficient to induce the cells to differentiate
(50). Retinoic acid-induced HL-60 cells have
morphological characteristics that correspond to normal
granulocytes and acquire the capacity to reduce NET and
produce 02". The induced cells also show positive
reactivity for chloroacetate esterase staining.

HL-60 cells have been found to have a highly
amplified c-myc oncogene (51,52). Westin et al (53)
have shown that, like in the teratocarcinoma F9 cell
line, the expression of the c-myc gene in HL-60 cells
is reduced 80-90% by RA during induction of
differentiation. Recently it was shown that 1,25-
dihydroxyvitamin D3 also suppresses the expression of
the c-myc oncogene in HL-60 cells (54) and that this
occurs prior to the onset of phenotypic changes. Even
though it appears that retinoids modify gene expression

during RA-induced differentiation, much more research

 

19

is needed to determine which genes are being altered

and the mechanisms involved.

D. Summary

Retinoids elicit many diverse effects on both
neoplastic and non-neoplastic cells, as can be seen in
the many examples cited above (see Table 13. Effects
of treatment with retinoids include: inhibition of
growth in monolayer, inhibition of anchorage-
independent growth in semi-solid medium and the
induction of terminal differentiation of neoplastic
cells to non-neoplastic differentiated cells. However,
it is important to note that the examples provided are
of specific cell types and that retinoids evoke a wide
range of responses in different cell types. Cell lines
from tumors of similar histopathology, as well as
subclones of a transformed cell line can often differ
in their response to retinoid treatment.

Different retinoids also elicit different
responses within a cell type. In many test syStems, RA
is at least 100 times more active than retinol and is
biologically active at very low concentrations. In
most cases, RA is also more active than retinyl esters
orretinaldehyde.

Growth inhibitory effects of retinoids can be seen

in many neoplastic and non-neoplastic cell lines and

20

Table I: Effects of Retinoic Acid on Growth Rate and
Differentiation of Several Cell Lines

 

 

 

Cell Line Diff. Growth Re:
3E

Non-Neoplastic

Human Epidermal Keratinocytes + 18,19

Mouse Mammary Gland - 21

Testicular Cells +/- 23

Adipocytes - NE 24,25

Chondrocytes - 26-28

Fibroblasts - 32

Neoplastic u

Embryonal Carcinoma Cells F9 + 35

Murine Melanoma Cells - 42

Human Promyelocytes HL-6O + - 13

Diff. = differentiation

Ref. = references

+ = stimulatory

- = inhibitory

NE no effect

II

21

can generally be reversed within 2-3 days after removal
of retinoid treatment. In certain cell systems,
retinoids can promote differentiation , while in others
it suppresses it. With the exception of terminally
differentiated systems, these effects are reversible
upon removal of the retinoid. Therefore, it is
important to realize that the effects of retinoids tend
to ibe cell-specific :making’ it. difficult to find a
common mechanism by which retinoids function in cell
proliferation and Cell differentiation. A single
common mechanism probably does not exist. In cell
culture studies, investigators tend to emphasize the
role of retinoids in either cell proliferation or cell
differentiation. At this point, retinoids appear to be
involved in both processes making it difficult to

separate the two.

REVIEW OF FUNCTION OF ALKALINE PHOSPHATASE

A. Physical and Chemical Properties of Alkaline

Phosphatase

Alkaline phosphatases are a group of membrane
bound glycoproteins with molecular weights generally in
the range of 100,000 to 200,000 (55). They are usually
dimers consisting of two similar subunits. Five major
AP isozymes have been identified in human tissues; they
have been grouped into 3 distinct protein classes: the
liver-kidney-bone group; the placental enzyme; and the
intestinal enzyme (56). The liver-kidney-bone group of
AP isozymes have similar’ mobilities upon. two-
dimensional electrophoresis after' desialylation, and
show essentially identical cross-reactivity (57). The
placental and intestinal isozymes have some cross-
reactivity with each other, but they differ in their
electrophoretic patterns (57). Alkaline phosphatases
are characterized by a broad substrate specificity with
alkaline pH optimum. The chemical reaction catalyzed
by AP is the transfer of a phosphoryl group from a
donor (very often R-O-HPO3") to an acceptor (R'-OH,

where R' is usually H).

22

23

AP
R-o-Hpo3‘ + R'-OH ————) R-OH + R'-o-HPo3'

Mg2+

The dimeric structure of AP has been shown to have
three pairs of ion-binding sites: two pairs with high

2+ and one pair with high affinity for

affinity for Zn
Mg2+ (58). These metal ion-binding sites and two seryl
residues which become phosphorylated and
dephosphorylated during catalysis, are part of the
active site of AP (58). The affinity of Pi for the
active site is strongly affected by pH and Zn2+. The

dissociation of Pi from the active site appears to be

the rate-limiting step.

B. Phosphoprotein Phosphatases and Alkaline

Phosphatases

Phosphoprotein phosphatases are a group of enzymes
which catalyze the reverse reactions of protein
kinases, enzymes which phosphorylate specific cellular
proteins during hormonal or growth factor stimulation
of target cells (59). Phosphorylation and
dephosphorylation of serine, threonine, or tyrosine

residues on enzymes and on regulatory proteins is an

24

important control mechanism for the regulation of many
biological processes (60). Many high molecular weight
phosphoprotein phosphatases can be converted to a Mr =
35,000 (35 K dal) subunit with its activity conserved
or enhanced. This 35 k dal subunit has intrinsic AP
activity, using p-nitrophenylphosphate as a substrate,
especially in those phosphoproteins phosphatases which
direct their activity towards phosphotyrosine residues

(60).

C. Protein Phosphorvlation/Dephosphorvlation in

Regulation of Growth and Differentiation

Tyrosine phosphorylation of proteins is believed
to be involved in the regulation of control pathways of
growth and differentiation, rather than in metabolic
processes (61). Richardson et al (62) recently showed
that when human chronic myelogenous leukemia K-562
cells were induced to differentiate through the
erythroid pathway, levels of Tyr-phosphorylated
proteins decreased. They, therefore, concluded that
the reduction in protein tyrosine phosphorylation is
correlated with differentiation.

Recently, Swarup et al (63) showed that AP
preparations from calf intestine, bovine liver

and. Escherichia coli (E. coli) selectively

25

dephosphorylate histones specifically phosphorylated at
tyrosine residues (phospho-Tyr-histones) and A-431
membrane proteins (phosphorylated at tyrosine) at low
enzyme concentrations. Other studies have since
confirmed. these findings (64-66). One ‘very recent
study (64) in particular, using 31P NMR spectroscopy,
investigated the kinetics of the dephosphorylation of
phospho-amino acids with AP from calf intestine or
E. 991i. Their results showed that the initial rates
of dephosphorylation of phospho-tyrosine (P-Tyr) and
phospho-serine (P-Ser) by AP were essentially the same
in a one-substrate system. However, in a two-substrate
system (P-Tyr and P-Ser), the ratio of the initial rate
for P-Tyr vs. P-Ser was 2.4 to 4.5, depending on pH and
buffer' conditions. This ratio illustates the
specificity of AP for P-Tyr over P-Ser at the free
amino acid level. .Alkaline. phosphatases may,
therefore, be a group of membrane bound glycoproteins
which represent a class of phosphoprotein phosphatases
that selectively dephosphorylate Tyr-phosphorylated
phosphoproteins. The structural resemblance between
P-Tyr residues and. p-nitrophenylphosphate, a
substrate of AP, supports this hypothesis (60).

Burch et al (66) demonstrated that AP from
embryonic skeletal cartilage (differentiated

Chondrocytes) dephosphorylates P-Tyr and P-Tyr-histones

l

 

26

at physiologic pH and that P-Tyr activity is likely an
intrinsic property of AP. Distribution of histologic
staining for AP showed that areas of cartilage with
elevated .AP activity' also had increased P-Tyr
phosphatase activity. Triiodothyronine stimulation
increased cartilage AP activity by 88% and also
increased P-Tyr phosphatase activity (106%). In vitro
incubation with known AP-inhibitors (vanadate,
levamisole, homoarginine) blocked both AP and P-Tyr
phosphatase activities. Cartilage AP dephosphorylated
P-Tyr-histones but had little to no activity when
P-Ser- or P-Thr-histones were used as substrates. The
authors concluded that AP functions as a phospho-
tyrosine phosphatase and may serve as a regulator of
Chondrocyte differentiation via protein
phosphorylation-dephosphorylation reactions.

Alkaline phosphatase has also been associated with
cellular differentiation in studies utilizing known AP
inhibitors (67,68). Detectable AP activity was fOund
to be localized in the outer plexiform layer (OPL) of
the developing chick retina on the 12th to 13th day of
incubation (67). Araki and Saito (67) injected the AP
inhibitor, levamisole, into embryonic eyes on either
day 13 or 15 of incubation. They found that injection
on day 13 caused various morphological alterations in

retinal development, including the appearance of

27

photoreceptor cells in the subretinal space, as well as
folding of both the outer plexiform and outer nuclear
layers. Degeneration of ganglion cells and thinning of
the nerve-fiber layer were also observed. Injection of
levamisole on day 15 did not significantly influence
retinal development. The authors suggested that AP
activity in the OPL of the developing chick retina may
be important for the onset of normal development of
synapses in the OPL and for the differentiation of the
photoreceptor cells. Lyaruu et al (68) investigated
the effects of another AP inhibitor,
l-p-bromotetramisole (pBTM), on the pyrophosphatase
activity of AP in mineralizing and non-mineralizing
hamster molar tooth germ cells in vitro.
1-p-Bromotetramisole, 10'3M, was cultured with three
day old first (M1) and second (M2) maxillary hamster
molar' germs for 2,8,12,16 and. 24 h. IHistological
examination. showed. that. the: M1 germs ‘were actively
mineralizing and the M2 were at an early proliferative
phase and unmineralizing. pBTM inhibited (17-26%) the
formation of trichloroacetic (TCA)-insoluble [32PJ-
phosphate from inorganic [32PJ-phosphate in the
proliferating M2 germ cells, but had no effect on the
actively mineralizing M1 germ cells. Addition of 10'5M
inorganic [32PJ-pyrophosphate in the culture medium

increased the inhibition of the formation of TCA-

 

28

insoluble [32P]-phosphate in the M2. pBTM almost
completely inhibited (93-100%) the formation of TCA-
insoluble [32P]-pyrophosphate in the non-mineralizing
M2 germs. In the actively mineralizing M1, pBTM
significantly inhibited (<50%) but did not completely
inhibit the formation of TCA-insoluble [32PJ-phosphate.
The authors concluded that the inhibition of the
pyrophosphatase activity by pBTM in the M2 germs
resulted in an increase of uncleaved pyrophosphate
which could not enter the cell and form TCA-insoluble
phosphorylated organic macromolecules inside. IIt was
suggested that the pyrophosphatase activity of AP is
involved in the phosphorylation and turnover of
macromolecules necessary for tooth germ cell

differentiation and proliferation.

D. Regplation of Alkaline Phosphatase bv Retinoids

 

A number of compounds have been implicated as
regulators of various tissue-specific APs (69)
including: glucocorticoids, cAMP, short chain fatty
acids and 1,25-dihydroxyvitamin D3 (69). Recent
studies have shown that vitamin A may also play a role
in the regulation of APs. Riley and Spearman (70)
showed that prolonged topical application of retinyl

acetate to the tail epidermis of mice caused increased

 

29

AP-positive bands separated by gel electrophoresis.
This observation suggests that vitamin A and its
metabolites :may influence .AP activity is some cell
types. Reese and his associates (6,7,71), in a series
of in vitro studies, showed that RA induces AP activity
in normal and neoplastic rat prostatic cells and in the
urothelium of the rat urinary bladder. It was found
that RA, at a concentration of 10 uM, increased AP
activity in both normal rat prostate cells and in the
Dunning R-3327 transplantable prostatic adenocarcinoma
in culture (6). In some clones of the R-3327 tumor,
the magnitude of increase over the control was 20 fold
during a 3 day period. Retinoic acid was found to be
3-4 fold more effective in inducing AP activity than
retinol or retinal. In one rapidly growing cell
line, UMS-1541Q, AP activity was barely detectable in
the uninduced state. After treatment with 10 uM RA,
increased. activity could. be detected. within 3-4 h.
This increase was totally blocked by actinomycin D and
cyclohexamide, suggesting' that. neW' mRNA. and. protein
synthesis is necessary for the increased activity of AP
induced by RA. In another study, Reese and Politano
(7) showed that the amount of AP activity per ug of DNA
in the urothelium of the rat urinary bladder decreased
70% over a 13 day culture period. Upon the addition of

10 uM RA, AP activity increased to normal levels during

30

a 3 day culture period. In a subsequent study by Reese
et al (71), cells derived from the Dunning R-3327 rat
prostatic adenocarcinoma were selected, .using a
p-nitrophenylphosphate-agarose overlay procedure, on
the basis of their inducibility for AP activity by RA.
Treatment of these cells with. RA (10 uM) did not
significantly affect their growth rate in log phase;
however, the saturation density was lowered to 40% that
of the controls. This effect on saturation density was
reversible 24 h after removing RA from the medium. RA-
treated cells were found to have a 35-40% greater cross
sectional area than controls, as measured by light-
scatter flow cytometry. This may account for the lower
saturation density. In a number of cells a correlation
has been observed between increased cell volume and
increased AP activity. In cultured hamster embryo
cells, the volume of cells with AP activity was found
to be twice that of cells without AP activity (72).
This effect has also been observed in HeLa cells, which
are a human cervical carcinoma cell line (73), and in
human embryonic intestinal cells (74). However, at
this time a cause-effect relationship between increased
cell volume and increased AP activity has not been
demonstrated.

Sato et al (8) determined if chemical compounds

reported to induce differentiation of leukemic cells,

31

also have differentiating effects on normal human
granulocytes, using AP activity as a marker. Of the 11
compounds they examined, only RA induced AP activity.
A dose-response effect on AP activity by RA and other
retinoids was observed; however, RA (lo'aM) was shown
to be the most potent inducer of AP activity. Cells
treated with RA were also treated either simultaneously
or 24 h after the addition of RA, with various
inhibitors affecting cell metabolism, such as
actinomycin D (1 ug/ml), cyclohexamide (5 ug/ml) and
hydroxyurea (2mM). The addition of actinomycin D and
cyclohexamide, inhibitors of RNA and protein synthesis,
respectively, suppressed AP induction by RA. This
indicates that denovo RNA and. protein synthesis is
necessary for the induction of AP activity by RA. In
contrast to this, the addition of hydroxyurea, an
inhibitor of DNA synthesis, had no effect on the RA-
induction of AP activity.

Adams and Melnykovych (10) showed that low
concentrations of retinol (10 rmI- 10 uM) elevated AP
activity in bovine endothelial cells in culture.
Retinoic acid and retinyl acetate was also shown to
induce AP activity at the concentrations examined.
Cell growth was not effected by any of the vitamin A
compounds. In the presence of both retinol and

dexamethasone, a synergistic stimulation of AP activity

32

was observed, implying different mechanisms of
induction.

It has been shown that RA induces differentiation
of F9 teratocarcinoma stem cells into parietal endoderm
(35). Strickland et al (37) found AP activity
increased. approximately' 1.5 fold. over' controls 'upon
treatment of these cells with RA (10'7M). In contrast
to this, more recent studies have demonstrated, both
histochemically and quantitatively, that AP activity is
decreased upon RA-induced differentiation of F9 cells
(10'6M RA) (75,76). Therefore, the use of AP as a
marker for differentiation of F9 teratocarcinoma cells
is questionable.

Other studies have also shown a decrease in AP
activity upon RA treatment of cultured cell lines
(77,78). Treatment of HeLa 65 cells with retinol
(10 uM) inhibited cortisol-induced AP activity after
72 h of treatment (77). Other vitamin A compounds had
similar effects. Treatment of a poorly differentiated,
human rectal adenocarcinoma cell line (HRT-18) with
3.3 x 10'5M RA caused a 10 fold decrease in AP
activity. The authors suggested that changes in AP
levels may not be a good marker of a more
differentiated phenotype in colonic cells. The above

described experiments illustate that AP is somehow

 

33

involved in cell differentiation, but at this time the

mechanism is not clear.

E. Summary

Alkaline phosphatases have been studied
extensively, but their physiological function(s) remain
unknown. It appears at this time that AP is a
phosphoprotein phosphatase and that it may be involved
in the growth regulatory cascade. Increased levels of
AP are associated with differentiated cell types.
Recently, it was shown that AP selectively hydrolyzes
tyrosine phophorylated phosphoproteins. The
phosphorylation of these proteins appears to be part of
the mechanism for signalling cells to undergo division.
Dephosphorylation of the tyrosine residues, possibly by
AP, inhibits cell division, allowing the cells to
differentiate.

In certain cell systems, retinoids have been shown
to stimulate AP activity, while in others it suppresses
it. This again illustrates that many of the diverse
responses retinoids evoke are cell-specific. It is
possible that RA-induced cell differentiation is
associated with alkaline phosphatase activity. The
objective of this study was to determine if RA-induced
differentiation of HL-60 cells is associated with

induction of AP activity.

MATERIALS AND METHODS

A. Material Sources

2-Amino-2-methyl-1-propanol, dexamethasone,
dimethyl sulfoxide (DMSO), napthol AS-D chloroacetate
esterase (kit 91-C), nitroblue tertrazolium (NBT),
p-nitrophenylphosphate (PNPP), nonidet P40, phorbol 12-
myristate-13-acetate (PMA) and trypan blue dye were
obtained from Sigma Chemical Company, St. Louis, MO.
Dulbecco's modified minimal essential medium (DMMEM),
RPMI 1640 medium, fetal bovine serum (FBS), and
penicillin- streptomycin solution were purchased from
Gibco, Grand Island, NY. Bovine serum albumin was

obtained from Armour Pharmaceutical Company, Kankakee,

IL. Percoll was obtained from Pharmacia Inc.,
Uppsala, Sweden. All-trans-retinoic acid
was purchased from Eastman Kodak Company,

Rochester, NY. 1,25-Dihydroxyvitamin D3 was a gift
from Dr. R. Simpson, University of Michigan, Ann Arbor,
MI.

All other chemicals were reagent grade.

34

35

B. Cells and Culture

HL-60 cells were provided by Dr. R. Simpson,
University of Michigan, Ann Arbor, MI and were
subcultured twice a ‘week. in RPMI 1640 media
supplemented with 10% heat inactivated FBS, penicillin
(100 units/ml) and streptomycin (100 ug/ml), at 37°C in
a humidified atmosphere of 5% C02 in air. Cell numbers
were estimated with a haemocytometer. Viability was
assessed by exclusion of trypan blue dye. Experiments
were conducted with cells in their midlogarithmic phase
of growth. Cells (1-4 x 105 cells/ml) were exposed to
all-trans-retinoic acid (lo-GM), dissolved in ethanol,
for O, l, 12, 24, 36, and 72 h and to
DMSO (180 mM), 1,25-dihydroxyvitamin D3 (10‘7M) and

dexamethasone (10'7M), prepared in ethanol, for 0,24

and 72 h. Control cells received only the vehicle
(ethanol). The final concentration of vehicle in media
was 0.1%. Control and treated cells were harvested by

centrifugation (850 g) for 10 min. at the indicated
times.

F9 teratocarcinoma cells were obtained from
American Type Culture Collection, Rockville, MD. Stock
cultures were maintained in uncoated plastic flasks in

DMMEM supplemented with 10% heat inactivited FBS and 1%

36

penicillin-streptomycin solution. Stocks were

subcultured by trypsinization every second day.
C. Assessment of Differentiation

Cell differentiation was assessed by NBT dye
reduction and positive reactivity for chloroacetate
esterase staining.

The ability to reduce NBT, in the development of
normal granulocytes, is at a low -level in
metamyelocytes and increases in banded and segmented
granulocytes (79). NET reduction was assayed as
previously described (46). Approximately 2 x 105
cells/ml were harvested by centrifugation, suspended in
0.2 ml of phosphate-buffered saline (PBS) containing
0.1% NET and 40 ng PMA and incubated 45 min. at 37°C.
At least 200 cells were examined in a haemocytometer
and the percentage of cells containing intracellular
blue-black formazan deposits was determined.

Specific esterase activity was determined using
napthol AS-D chloroacetate as a substrate. Microscope
slides of cells were prepared and allowed to dry at
least 1 h prior to fixation. Cells were fixed for 30
sec. at room temperature (20-220C) in a citrate-
acetone-formaldehyde solution containing 25 ml of
citrate solution (composed of 18mM citric acid, 9mM

sodium citrate and 12mM NaCl), 65 ml of acetone and 8ml

 

37

of 37% formaldehyde. To 1 ml of fast red violet LB
base solution (15 mg/ml fast red violet LB base in 0.4M
HCL) 1 ml of sodium nitrite (0.1M) was added, vortexed
vigorously for 30 sec. and then added to 40 ml of
prewarmed (37°C) deionized water. Trizmal buffer
concentrate, 5 ml, (Trizma maleate 1M, pH 6.3) was
added, followed by 1 ml of napthol AS-D chloroacetate
solution (8 mg/ml). Slides were incubated in this
solution for 15 min. at 37°C protected from light.
After 15 min. the slides .were _removed and rinsed
thoroughly in deionized water. The slides were then

counter-stained for 2 min. in hematoxylin solution

(0.6 g/l sodium iodate and 52.8 g/l aluminum
sulfate). The slides were then rinsed in tap water
and air dried. The slides were evaluated
microscopically at x100 magnification. Sites of

activity were identified by the presence of bright

red granulation.

D. Isolation of Human Granulocytes

As a positive control, normal human granulocytes
were isolated by the method of Jepson and Skottun (80)
from fresh whole blood, which was provided by Dr. A.
Dimitrov, Michigan State University, East Lansing, MI.
In this method granulocytes can be separated from

monocytes and erythrocytes using two different

38

gradients of Percoll (density solution I: 1.075 Kg
Percoll/1; solution II: 1.096-1.098 Kg Percoll/l).
Percoll was diluted directly with 1.5 M NaCl to
obtain final working solutions of the above densities.
The densities and corresponding quantities were
calculated on the basis of figures given by Pharmacia
(81). Solutions I and II were prepared by weighing in
order to minimize variations in the densities.
Heparinized fresh venous blood, 8 ml, was diluted 1:2
with 0.15M NaCl. Four ml were distributed into each
test tube. Solution I, 3 ml, was placed underneath the
blood. Then 3 ml of solution II was carefully placed
beneath solution I. The test tubes were then
centrifuged at 200 g for 25 min. at room temperature
(20-22°C). Two separate bands formed which correspond
to the interphases. The upper band was made of
mononuclear cells, the lower band of granulocytes; the
pellet consisted of erythrocytes. The lower band was
removed with a wet pipette and transferred to washing
medium (PBS). Washing was conducted at 4°C and 800 g

for 10 min. and was repeated if necessary.

E. Preparation of Intestinal Mucosa

The intestinal absorbtive epithelium is.e1 rich

source of AP. Young adult male Sprague-Dawley rat

39

small intestinal mucosal cells were isolated to
provide another positive control. Small intestine was
removed, slit lengthwise and rinsed free of debris with
PBS, pH 7.2 . The intestine was then placed on a cold
plate and mucosa was scraped off, weighed and placed in
3-4 ml PBS. The mucosa was then homogenized using a

polytron (Brinkman Instruments, Westbury, N.Y. 11590).

F. Alkaline Phophatase Assay

For determination of alkaline phosphatase activity
(82), cells were washed with PBS and lysed by a series
of freeze-thaw cycles and by ‘the addition of 0.2%
nonidet P40. Alkaline phosphatase activity was
measured by incubating aliquots of cell lysates
(10-50 ug protein) at 37°C in 0.5 M 2-amino-2-methyl-1-
propanol buffer, pH 10.3 containing 1 mM MgClz, with
8 mM PNPP as the substrate for 30 min. in a shaking
water bath. The final reaction volume was 1.5 ml. The
reaction was terminated by the addition of an equal
volume of 0.2 N NaOH. The amount of p-nitrophenol
(PNP) released was measured spectrophotometrically at
410 nm. The reaction was linear for 30 min. Alkaline
phophatase activity was expressed as nmoles PNP

liberated/ 30 min./ mg protein.

40

G. Protein Assay

Protein concentrations were estimated by a
modified Lowry procedure (83), using bovine serum

albumin as standard.

 

 

RESULTS

A. Treatment of HL-60 Cells with Retinoic Acid

Alkaline phosphatase activity was not
detectable in HL-60 cells prior to or after treatment
with RA (10'6M) for 72 h. Under these conditions, 50-
70% of the cells differentiated as evidenced by their
ability to reduce NBT (Figs. 1,6) and the presence of

specific esterase activity (Fig. 2).

B. Control Experiments with Cells Known to have

Alkaline Phosphatase Activity

Several positive control experiments were
conducted in order to determine if the assay was a
sensitive indicator of AP activity at the protein
levels used in assaying the HL-60 cells. Cells known
to have AP activity, such as F9 teratocarcinoma cells,
rat intestinal mucosa and normal human granulocytes,
were assayed at protein concentrations from 10 ug to
1. mg. 'Fhe :results obtained, as shown. in Fig. 3,
indicate that the assay can. detect AP {activity at
protein levels as low as 10 ug (Fig. 3 inset).

However, at protein levels as high as 630 ug, AP

41

 

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44

Napthol AS-D Chloroacetate Esterase Activity in
Retinoic Acid Treated HL—6O Cells

Control cells (A,B) demonstrate by light microscopy
(magnification x100), (A) sites of activity by the
presence of bright red granulation in normal

human granulocytes and (B) the absence of activity

in ethanol-treated HL-6O controls. RA-treated HL-60
cells (C), show the presence of chloroacetate esterase

activity after 72 h of treatment.

45

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activity was still not detectable in HL-60 cells either

prior to or after induction of differentiation.

C. Effect of Known Inducers on Alkaline Phosphatase

Activity in HL-60 Cells

The next set of experiments were conducted to
determine if: 1) AP was present in HL-60 cells but was
not inducible with RA; In .AP could be induced with
known inducers of AP activity, such as 1,25-
dihydroxyvitamin D3 and dexamethasone; and 3) AP
could be induced by other differentiating agents, such
as 1,25-dihydroxyvitamin D3 and DMSO, which cause HL-60
cells to differentiate towards the* monocyte and
granulocyte cell types, respectively. HL-60 cells were
incubated ‘with 1,25-dihydroxyvitamin D3, 10'7M, and
dexamethasone, 10-7M, and DMSO, 180 mM. Alkaline
phosphatase activity could not be detected with any of
the above compounds (Table II). Figures 4-6 illustrate
the extent of differentiation after 72 h of continuous
treatment with various agents.
Treatment with 1,25—dihydroxyvitamin D3 (Fig. 4)
(72 h), an inducer of AP activity and a differentiating
agent, differentiated 71.1% of the cells, while only 5%
of the control cells which received the vehicle,

ethanol (<1.0%), were differentiated. Dexamethasone

 

49

Table II: Alkaline Phosphatase Activity in HL-60 Cells
Following Treatment with Inducers

AP Activity
nmoles PNP liberated/ 30 min./ mq Prot.

 

Vehicle (ethanol < .0%) ND
Retinoic Acid (10' M) ND
1,25-dihydroxyvitamin D3 (10'7M) ND
Dimethylsulfoxide_(180mM) ND
Dexamethasone (10 M) ND
AP alkaline phosphatase

PNP
ND

p-nitrophenol
not detectable

 

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DISCUSSION

Retinoic acid induces differentiation in many

cells, and this differentiation generally results in a
more mature cell type and is associated with the
appearance of biochemical markers characteristic of the
newly differentiated phenotype. Retinoic acid induces
differentiation of promyelocytic HL-60 cells (13) into
more mature granulocytic precursors. These induced
'cells have many functional characteristics of normal
human peripheral granulocytes, including phagocytosis,
lysozomal enzyme release, hexose monophosphate shunt
activity, the generation of.superoxide anion (02') and
the ability to reduce NBT (45-47). Sato et al (8)
showed that AP activity was induced by RA in
postmitotic granulocytes from bone marrow of normal
human donors. They suggested that the RA-induction of
AP activity was a marker for normal granulocytic
differentiation and maturation. Stewart (84) showed
that the myelocyte was the earliest myeloid cell to
‘ ShOW’ AP activity' and that .AP levels increase with
nuclear maturation. It was therefore logical to ask if
RA-induced differentiation of HL-60 cells is associated
with the induction of AP activity. The induction of AP

was of particular interest since dephosphorylation of

57

.-....

 

 

58

growth regulatory proteins is an important mechanism to
down-regulate cell proliferation and tx: generate
signals for cell differentiation. Alkaline
phosphatase, an ubiquitous enzyme with poorly defined
physiological functions, is generally associated with
highly differentiated cells and could conceivably be an
important modulator of this process.

Alkaline phosphatase activity was not
detectable in HL-60 cells prior to or after treatment
with RA or other inducers of both differentiation or AP
activity. A number of positive control experiments
were conducted in order to validate the enzyme assay
conditions. The values obtained for AP activity are
similar to those reported in the literature. F9
teratocarcinoma cells were found to have 2.5 umol PNP/
30 min./ mg protein. Under similar conditions,
Berstine et al (85) reported a range of 2.5-14.7 umol
PNP/ 30 min./ mg protein for F9 cells. Komoda et al
(86) reported adult rat intestinal mucosal AP activity
levels at approximately 1.1 umol PNP/ 30 min./ mg
protein. In this study an activity of 1.7 umol/ 30
min. / mg’ protein was obtained for rat intestinal
mucosa. Rosenblum and Petzold (87) obtained 660 nmol
PNP/ 30 min./ mg protein from. peripheral blood of
normal donors. In this study normal granulocytes, also

isolated from peripheral blood of normal human donors,

 

59

had 811 nmol PNP/ 30 min./ mg protein. The results
indicate that the assay was functioning similarly to
assays conducted in other laboratories. In summary,
this study clearly demonstrates that the assay system
for AP was valid and sensitive to detect the presence
of AP.

The results indicate that the ability to
synthesizeiAP, which is gained as normal granulocytes
mature from promyelocytes to myelocytes, is not
developed during RA-induced differentiation of HL-60
cells. Therefore, RA-induced differentiated HL-60
cells may be at a stage before the more mature function
of AP is expressed. Similarly, in the case of 1,25-
dihydroxyvitamin D3 and DMSO, differentiating agents
that cause promyelocytic HL-60 cells to differentiate
into monocyte-l ike and granulocyte-l ike cel ls ,
respectively, there was no induction of AP. It is not
known, however, if normal human monocytes have AP; and
therefore it is not possible to compare the results
that were obtained with the monocytes derived from
differentiated HL-60 cells. On the other hand it may
be that AP is not present in HL-60 cells and that this
may be the reason that these cells are an immortal
cell line. This appears to be a more logical
explanation because not only did the differentiating

agents not induce AP, but known AP inducers,

 

navy-

 

60

such as dexamethasone , also had no effect on the
HL-60 cells. HL-60 cells may not have AP to
dephosphorylate a growth regulatory protein in order to
cease cell division. In either case, it is clear that
RA-induced differentiation of HL-60 cells occurs
through a mechanism that does not involve AP.

Several mechanisms of RA-induced differentiation
have been proposed. Cellular retinoic acid-binding
protein (CRABP) and cellular retinol-binding protein
(CRBP) have been detected in the cytosol of several
normal tissues and tumor cell lines (2). Many studies
suggest that these binding proteins mediate the
biological activities of retinoids of growth and
differentiation (25,88). However, CRABP has not been
detected in HL-60 cells, suggesting that RA-induced
differentiation of HL-60 cells is not mediated by
retinoid-binding proteins (89). Some evidence suggests
that. different protein. kinases are involved in. the
regulation of differentiation of HL-60 cells (90). The
amplified expression of the c-myc oncogene is reduced
during RA-induced differentiation (53). Therefore, it
appears that retinoids may modify gene expression
during RA-induced differentiation; Evidence has also
been presented for a coenzyme A mediated activation of
RA (91). At this point the mechanism of RA-induced

differentiation of HL-60 cells is not known. It may be

 

61

that the recently identified nuclear receptor for
retinoic acid (92) accounts for the function of RA in
HL-60 cells. A single mechanism probably does not
exist. Further studies may lead to a better
understanding of the mechanisms involved in retinoid

modulation of cellular growth and differentiation.

 

CONCLUSIONS

Alkaline phosphatase activity was not detectable
in the human promyelocytic cell line HL-60 either prior
to or after 'treatment with retinoic acid or other
inducers of differentiation and AP activity. These
results suggest that RA-induced HL-60 cells may not be
at a stage of’ maturation at which. AP activity is
expressed or that AP activity is not present in HL-60
cells. :rt appears that RA-induced differentiation of
HL-60 cells occurs through a mechanism that does not

involve alkaline phosphatase.

62

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