G-CSF

Shigekazu Nagata*
Department of Genetics, Osaka University Medical School, 2-2 Yamada-oka Suita,
Osaka, 565-0871, Japan
* corresponding author tel: 81-6-6879-3310, fax: 81-6-6879-3319,
e-mail: nagata@genetic.med.osaka-u.ac.jp
DOI: 10.1006/rwcy.2000.09006.

SUMMARY
G-CSF is a 2025 kDa glycoprotein that specifically
regulates the production of neutrophilic G granulo-
cytes as well as enhancing the functional activities of
mature neutrophils. It is produced by activated macro-
phages, endothelial cells, and fibroblasts. G-CSF is
widely used clinically in the treatment of patients with
neutropenia after cancer chemotherapy.
BACKGROUND
Discovery
G-CSF (granulocyte colony-stimulating factor) was
discovered in serum from endotoxin-treated mice as a
factor that stimulates neutrophilic colony formation
from bone marrow cells, and that induces differentia-
tion of mouse WEHI-3B D cells into neutrophilic
granulocytes (Nicola et al., 1983). Some human cell
lines such as squamous carcinoma CHU-2 and blad-
der carcinoma 5637 were found to constitutively pro-
duce G-CSF (Welte et al., 1985; Nomura et al., 1986),
and its cDNA was cloned from these cell lines
(Nagata et al., 1986a,b; Souza et al., 1986).
Subsequently, mouse G-CSF was identified from a
mouse NFSA cDNA library by crosshybridization
with human cDNA (Tsuchiya et al., 1986).
Alternative names
G-CSF has also been known as colony-stimulating
factor 3 (CSF-3), macrophage and granulocyte inducer
type 1, granulocyte (MGI-1G), granulocyte-macro-
Structure
G-CSF is a 2025 kDa glycoprotein.
Main activities and
pathophysiological roles
G-CSF regulates production of neutrophilic granulo-
cytes, and activates mature neutrophils.
GENE AND GENE REGULATION
Accession numbers
Human G-CSF: M13008 (Nagata et al., 1986a,b;
Souza et al., 1986)
Murine G-CSF: M13926 (Tsuchiya et al., 1986)
Chromosome location
Human chromosome 17q21-q22 (Kanda et al., 1987)
Mouse chromosome 11 (Buchberg et al., 1988)
Regulatory sites and corresponding
transcription factors
There are three regulatory sequences (GPE, G-CSF
promoter element) in the 300 bp upstream from the
transcription initiation site. GPE1 contains cis-
elements for NFB and NF-IL6, while GPE2 is a
phage colony-stimulating factor  (GM-CSF), and typical cis-element for OCT (octamer) transcription
pluripotent colony-stimulating factor (pluripoietin). factor (Nishizawa and Nagata, 1990; Nishizawa et al.,
936 Shigekazu Nagata

1990). Accordingly, fibroblasts from NF-IL6-defi- Important homologies

cient mice do not produce G-CSF upon stimulation
by IL-6 or TNF.
Cells and tissues that express
the gene
The G-CSF gene is expressed in monocytes, macro-
phages, endothelial cells, and fibroblasts.
Human and mouse G-CSFs are 73.6% identical at the
amino acid sequence level (Tsuchiya et al., 1986).
There is a significant sequence homology between
G-CSF and IL-6 (Hirano et al., 1986). The tertiary
structure of human G-CSF is similar to those of IL-4,
IL-2, and growth hormone although the similarity in
the primary sequence is not significant (Hill et al.,
1993; Lovejoy et al., 1993; Zink et al., 1994).

Posttranslational modifications
PROTEIN
Human G-CSF is O-glycosylated at Thr133. The
Accession numbers structure of the sugar moiety attached to human G-
Human G-CSF: PID g117564; SwissProt P09919
(Nagata et al., 1986a,b; Souza et al., 1986)
Mouse G-CSF: PID g117565; SwissProt P09920
(Tsuchiya et al., 1986)
Sequence
See Figure 1.
Description of protein
The N-terminal 30 amino acids serve as a signal
sequence for secretion. Cys36 and Cys42, as well as
Cys67 and Cys77, are connected by disulfide bonds
(Lu et al., 1989). The isoelectric point of the protein is
5.56.1, depending on the degree of sialylation
(Nomura et al., 1986). It is relatively stable to
extreme pH levels (pH 2 or pH 10), temperature (50%
loss of the activity at 70 C for 30 min), and strong
denaturation agents (6 M guanidine hydrochloride,
8 M urea, 0.1% SDS) (Nicola et al., 1983).
Discussion of crystal structure
G-CSF has a four  helix bundle structure (Hill et al.,
1993; Lovejoy et al., 1993; Zink et al., 1994).
Figure 1 Amino acid sequence for human G-CSF.
CSF is N-acetylneuraminic acid (2-6)[galactose (1-
3)] N-acetylgalactosamine (Souza et al., 1986; Oheda
et al., 1990).
CELLULAR SOURCES AND
TISSUE EXPRESSION
Cellular sources that produce
Monocytes, macrophages, endothelial cells, and
fibroblasts are induced to express G-CSF by various
stimuli (Metcalf and Nicola, 1985; Broudy et al.,
1987; Koeffler et al., 1987; Seelentag et al., 1987;
Kaushansky et al., 1988; Lu et al., 1988; Vellenga
et al., 1988; Nishizawa and Nagata, 1990). Some
carcinoma cells, such as human squamous carcinoma
CHU-2, bladder carcinoma 5637, glioblastoma
U87MG, and hepatoma SK-HEP-1 cell lines produce
G-CSF constitutively (Nomura et al., 1986; Tweardy
et al., 1987).
Eliciting and inhibitory stimuli,
including exogenous and
endogenous modulators
TNF as well as IL-1 stimulate fibroblasts or
endothelial cells to produce G-CSF (Broudy et al.,
 G-CSF 937

1987; Koeffler et al., 1987; Seelentag et al., 1987; also assayed by its ability to induce differentiation of
Kaushansky et al., 1988). Endotoxins stimulate WEHI-3B D or 32D cells into neutrophils.
macrophages to produce G-CSF (Metcalf and Nicola,
1985; Nishizawa and Nagata, 1990).
IN VIVO BIOLOGICAL
ACTIVITIES OF LIGANDS IN
RECEPTOR UTILIZATION ANIMAL MODELS
G-CSF has a unique receptor (G-CSF receptor).
Normal physiological roles
The normal physiological role of G-CSF is the produc-
IN VITRO ACTIVITIES tion of neutrophils.

In vitro findings
Species differences
G-CSF stimulates the colony formation of neutro-
philic granulocytes in semi-solid cultures of bone G-CSF has no species-specificity between human and
marrow cells (Nicola et al., 1983). Unlike other CSFs mouse (Tsuchiya et al., 1986).
such as GM-CSF and IL-3, G-CSF is rather specific
to progenitor cells of neutrophilic granulocytes. G-
CSF stimulates not only proliferation and differentia-
tion of the progenitors, but also prolongs the survival Knockout mouse phenotypes
of the mature neutrophils and enhances the functional
capacity of the mature neutrophils (Kitagawa et al., Mice lacking the G-CSF gene show chronic
1987; Williams et al., 1990; Yuo et al., 1989). Several neutropenia, are deficient in granulocyte and macro-
myeloid leukemia cell lines such as mouse NFS60 and phage progenitor cells, and show impaired neutrophil
human TF-1 cells proliferate in response to G-CSF mobilization (Lieschke et al., 1994).
(Tsuchiya et al., 1986; Kitamura et al., 1989), whereas
some other myeloid cell lines, such as mouse WEHI-
3B D, 32D, and L-G, can be induced to differentiate Transgenic overexpression
into neutrophilic granulocytes by G-CSF (Nicola
et al., 1983; Valtieri et al., 1987; Lee et al., 1991). Long-term exposure of mice to G-CSF in transgenic
mice causes sustained granulocytosis (Chang et al.,
1989).

Regulatory molecules: Inhibitors

and enhancers Pharmacological effects
G-CSF-induced neutrophilic colony formation from Administration of G-CSF into mice stimulates
bone marrow is inhibited by IFN , lymphotoxin, and granulopoiesis (Cohen et al., 1987; Tsuchiya et al.,
TNF (Barber et al., 1987). 1987; Welte et al., 1987).

Bioassays used PATHOPHYSIOLOGICAL ROLES

IN NORMAL HUMANS AND
G-CSF can be assayed by its neutrophilic colony-
stimulating activity in semi-solid culture of bone DISEASE STATES AND
marrow cells. In this assay, G-CSF has a specific DIAGNOSTIC UTILITY
activity of about 2  108 units/mg protein (where
50 units/mL is the concentration required for half- Normal levels and effects
maximal stimulation). G-CSF can be assayed by the
MTT method, or [3H]thymidine incorporation into Serum of healthy persons contains less than 30 pg/mL
G-CSF-responsive cells such as NFS-60 cells. It is of G-CSF. Its level increases to 502000 g/mL in
938 Shigekazu Nagata
patients with acute bacterial infections. The G-CSF
levels rise during the neutropenic phase of cyclic
neutropenia (Watari et al., 1989).
IN THERAPY
Preclinical How does it affect
disease models in animals?
Administration of G-CSF protects neutropenic mice
from lethal bacterial infection by accelerating
recovery of neutrophils (Matsumoto et al., 1987).
Effects of therapy: Cytokine,
antibody to cytokine inhibitors, etc.
G-CSF is effectively used to stimulate granulopoiesis
in neutropenic patients.
Pharmacokinetics
The distribution half-life is about 30 minutes and the
elimination half-life is about 3.8 hours in mice (Cohen
et al., 1987). The elimination half-life in patients
varies from less than an hour to several hours with
some evidence that this is dependent on neutrophil
numbers and receptor-mediated clearance (Watari
et al., 1997).
Toxicity
No severe toxicity.
Clinical results
G-CSF is widely used clinically in patients with
granulopenia from various causes (Ganser and
Karthaus, 1996; Welte et al., 1996). G-CSF is admin-
istered to patients with cancer receiving chemo-
therapy or radiotherapy with or without bone marrow
transplantation, and to patients receiving immuno-
suppressive agents after organ transplantation. In
both types of patients, G-CSF accelerates the
recovery of neutrophilic granulocytes and diminishes
the risk of severe bacterial and fungal infections after
chemotherapy.
In addition, G-CSF is currently used to mobilize
hematopoietic progenitor cells out of the bone
marrow into the blood allowing autologous bone
marrow transplantation (BMT) to be replaced by
peripheral blood stem cell (PBSC) transplants. This
procedure allows more rapid hematopoietic recovery
than that seen by traditional BMT (Sheridan et al.,
1992).
G-CSF has also been used to treat patients with
cyclic neutropenia. While it does not alter the cyclic
nature of this disease, G-CSF elevates neutrophil
levels during the nadir phase, thus preventing many of
the symptoms of the disease (Hammond et al., 1989).
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LICENSED PRODUCTS
Neutrogen1, recombinant human G-CSF produced
in CHO cells, is available from Chugai Pharma-
ceutical Co., Tokyo, Japan.
Neupogen1 (Filgrastim), recombinant human G-CSF
produced in E. coli, is available from Amgen,
Thousand Oaks, California, USA.