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Advances in
CANCER
RESEARCH
Volume 80

This Page Intentionally Left Blank

Advances in
CANCER
RESEARCH
Volume 80
Edited by
George F. Vande Woude
Division of Basic Sciences
National Cancer Institute
National Institutes of Health
Bethesda, Maryland
George Klein
Microbiology and Tumor Biology Center
Karolinska Institutet
Stockholm, Sweden
San Diego San Francisco New York Boston London Sydney Tokyo

Contents
Contributors to Volume 80 ix
Involvement of Platelet-Derived Growth Factor in Disease:
Development of Speciﬁc Antagonists
Arne Östman and Carl-Henrik Heldin
I. Introduction 2
II. Platelet-Derived Growth Factor (PDGF) and PDGF Receptors 3
III. Intracellular Signal Transduction 5
IV. In Vivo Function of PDGF 9
V. PDGF in Disease 13
VI. PDGF and PDGF Receptors as Drug Targets 19
VII. Future Perspectives 27
References 28
Tumor Suppression Activity of Adenovirus E1a Protein:
Anoikis and the Epithelial Phenotype
Steven M. Frisch
I. Introduction 39
II. Historical Development of E1a as a Tumor Suppressor 40
III. Epithelial Conversion: Phenomenology 41
IV. Epithelial Conversion: Mechanisms 41
V. Anoikis Sensitization and Tumor Suppression by E1a 44
References 46
Comparative Analysis of the Transforming Mechanisms
of Epstein–Barr Virus, Kaposi’s Sarcoma-Associated
Herpesvirus, and Herpesvirus Saimiri
Blossom Damania and Jae U. Jung
I. Introduction 51
v

II. Comparative Analysis of Gamma Herpesvirus Gene Products 55
III. Conclusion 73
References 74
Genetic Predisposition and Somatic Diversification
in Tumor Development and Progression
Darryl Shibata and Lauri A. Aaltonen
I. Introduction 84
II. Hereditary Colorectal Cancer Syndromes 85
III. Tumor Mutations and Molecular Clocks: Gateways to the Fourth Dimension 89
References 110
Primary Effusion Lymphoma: A Liquid Phase Lymphoma
of Fluid-Filled Body Cavities
Gianluca Gaidano and Antonino Carbone
I. Definition of Primary Effusion Lymphoma 116
II. Histogenes of Primary Effusion Lymphoma 119
III. Pathogenesis of Primary Effusion Lymphoma 121
IV. Epidemiology of Primary Effusion Lymphoma 133
V. Clinical Features of Primary Effusion Lymphoma 135
VI. Radioimaging of Primary Effusion Lymphoma 136
VII. Differential Diagnosis of Primary Effusion Lymphoma 136
VIII. Therapy of Primary Effusion Lymphoma 137
IX. Perspectives 138
References 140
Dimensions of Antigen Recognition and
Levels of Immunological Specificity
Neil S. Greenspan
I. Introduction 148
II. Logical Preliminaries: Boundaries of Categories and Categories
of Boundaries 149
III. Monovalent Recognition 153
IV. Multivalent Recognition 165
V. Specificity of Cellular Activation 171
VI. Organismal Specificity 174
VII. Conclusions 182
References 183
vi Contents

Topoisomerase I-Mediated DNA Damage
Philippe Pourquier and Yves Pommier
I. Introduction 189
II. Structural Domains of Top1 190
III. Top1 Functions and Protein Interactions 191
IV. The Top1 Catalytic Cycle and Cleavage Complexes 195
V. Anticancer Top1 Poisons 198
VI. Suppression and/or Enhancement of Top1 Cleavage Complexes
by DNA Damage 201
VII. Processing of Top1-Mediated DNA Lesions 209
VIII. Conclusions 211
References 211
Index 217
Contents vii

Contributors
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
Lauri A. Aaltonen, Department of Medical Genetics, Haartman Institute,
University of Helsinki, FIN-00014 Helsinki, Finland (83)
Antonino Carbone, Division of Pathology, Centro di Riferimento Oncologi-
co, IRCCS, Istituto Nazionale Tumori, 33081 Aviano, Italy (115)
Blossom Damania, Department of Microbiology and Molecular Genetics,
New England Regional Primate Research Center, Harvard Medical
School, Southborough, Massachusetts 01772 (51)
Steven M. Frisch, The Burnham Institute, La Jolla, California 92037 (39)
Gianluca Gaidano, Division of Internal Medicine, Department of Medical
Sciences, Amedeo Avogadro University of Eastern Piedmont, 28100 No-
vara, Italy (115)
Neil S. Greenspan, Institute of Pathology, Case Western Reserve University,
Cleveland, Ohio 44106 (147)
Carl-Henrik Heldin, Ludwig Institute for Cancer Research, Biomedical Cen-
ter, S-751 24 Uppsala, Sweden (1)
Jae U. Jung, Department of Microbiology and Molecular Genetics, New En-
gland Regional Primate Research Center, Harvard Medical School, South-
borough, Massachusetts 01772 (51)
Arne Östman, Ludwig Institute for Cancer Research, Biomedical Center,
S-751 24 Uppsala, Sweden (1)
Yves Pommier, Laboratory of Molecular Pharmacology, Division of Basic
Sciences, National Cancer Institute, National Institutes of Health, Bethes-
da, Maryland 20892 (189)
Philippe Pourquier, Laboratory of Molecular Pharmacology, Division of
Basic Sciences, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892 (189)
Darryl Shibata, Department of Pathology, Norris Cancer Center, University
of Southern California School of Medicine, Los Angeles, California 90033
(83)
ix

Involvement of Platelet-Derived
Growth Factor in Disease:
Development of Speciﬁc Antagonists
Arne Östman and Carl-Henrik Heldin
Ludwig Institute for Cancer Research
Biomedical Center
S-751 24 Uppsala, Sweden
I. Introduction
II. Platelet-Derived Growth Factor (PDGF) and PDGF Receptors
A. PDGF Isoforms
B. Cell Types Producing PDGF
C. Processing and Compartmentalization of PDGF
D. PDGF Receptors
E. Target Cells of PDGF
F. Cellular Effects of PDGF Isoforms
III. Intracellular Signal Transduction
A. Receptor Activation
B. Signaling via SH2 Domain Proteins
C. Modulation of Signaling
IV. In Vivo Function of PDGF
A. Embryonal Development
B. Wound Healing
C. Vascular System
D. Tissue Homeostasis
V. PDGF in Disease
A. Cancer
B. Atherosclerosis
C. Lung Fibrosis
D. Glomerulonephritis
VI. PDGF and PDGF Receptors as Drug Targets
A. PDGF Antagonists
B. PDGF Antagonists in Treatment of Cancer
C. PDGF Antagonists in Treatment of Atherosclerosis
D. PDGF Antagonists in Treatment of Lung Fibrosis
E. PDGF Antagonists in Treatment of Glomerulonephritis
VII. Future Perspectives
References
Platelet-derived growth factor (PDGF) is a family of dimeric isoforms that stimulates,
e.g., growth, chemotaxis and cell shape changes of various connective tissue cell types
Advances in CANCER RESEARCH Vol. 80 Copyright © 2001 by Academic Press.
0065-230X/01 $35.00 All rights of reproduction in any form reserved.

and certain other cells. The cellular effects of PDGF isoforms are exerted through bind-
ing to two structurally related tyrosine kinase receptors. Ligand binding induces recep-
tor dimerization and autophosphorylation. This enables a number of SH2 domain con-
taining signal transduction molecules to bind to the receptors, thereby initiating various
signaling pathways. PDGF isoforms have important roles during the embryonic devel-
opment, particularly in the formation of connective tissue in various organs. In the adult,
PDGF stimulates wound healing. Overactivity of PDGF has been implicated in certain
disorders, including fibrotic conditions, atherosclerosis, and malignancies. Different
kinds of PDGF antagonists are currently being developed and evaluated in different an-
imal disease models, as well as in clinical trials. © 2000 Academic Press.
I. INTRODUCTION
Accumulating evidence supports the notion that loss of growth control of
cancer cells involves perturbation of signaling pathways that in the normal
cell are controlled by growth regulatory factors. Thus in cancer cells, growth
stimulatory factors may be produced that stimulate cell growth in an au-
tocrine manner, growth factor receptors may be amplified or mutated so that
they are also active in the absence of stimulation by growth factor, or intra-
cellular components, which in the normal cell are controlled by growth fac-
tors, may be overactive. Analogously, growth inhibitors that normally con-
trol the growth of a particular cell type may be lacking, or receptors for
growth inhibitors or their downstream effectors may be defect or absent. In
each case the balance between stimulatory and inhibitory signals is altered,
and loss of growth control results.
Platelet-derived growth factor (PDGF) is a connective tissue cell mitogen
that has been implicated in tumorigenesis. Its transforming properties were
illustrated by the findings that the gene for one of the PDGF isoforms was
acquired as the sis oncogene of the simian sarcoma virus (SSV) and that
SSV transformation of cells involves autocrine stimulation by a PDGF-like
growth factor (reviewed by Westermark et al., 1987). PDGF has also been
implicated in autocrine as well as paracrine mechanisms in various types of
human cancers, as well as in other diseases characterized by an excessive cell
growth (reviewed by Heldin and Westermark, 1999).
The signaling mechanisms whereby PDGF exerts its cellular effects and the
in vivo function and role in disease of PDGF have been reviewed (Betsholtz
and Raines, 1997; Heldin and Westermark, 1999; Heldin et al., 1998; Ro-
senkranz and Kazlauskas, 1999). The aim of this review is to briefly update
the progress in our understanding of PDGF signaling and the role of PDGF
in vivo and in diseases and to focus on the design and development of PDGF
antagonists and discuss their potential clinical use.
2 Arne Östman and Carl-Henrik Heldin

II. PDGF AND PDGF RECEPTORS
PDGF is a family of isoforms that exerts their cellular effect by binding to
two structurally related tyrosine kinase receptors.
A. PDGF Isoforms
PDGF family members are dimeric molecules with a conserved sequence
of about 100 amino acid residues containing a characteristic motif of 8
cysteine residues. Three isoforms, i.e., homo- or heterodimers of A and B
polypeptide chains, are well characterized (reviewed by Heldin and Wester-
mark, 1999). Interestingly, however, another PDGF isoform has been dis-
covered, i.e., PDGF C (Li et al., 2000). The C chain occurs as a homodimer
(PDGF CC); it is not known whether it can also form heterodimers with oth-
er PDGF chains. Moreover, PDGFs are structurally similar to the vascular
endothelial growth factor (VEGF) family (VEGF, VEGF-B, VEGF-C, and
VEGF-D and placenta growth factor; reviewed in Joukov et al., 1997).
Crystallization of PDGF BB revealed that the two subunits in the dimer are
arranged in an antiparallel manner (Oefner et al., 1992). Two cysteine resi-
dues form interchain disulfide bonds. The other six conserved cysteine resi-
dues are arranged in a tight knot structure with one disulfide bond passing
through the hole formed by the other two disulfide bonds and intervening
amino acid residues. Two loops (loop 1 and loop 3) extend in one direction
from the cystine knot and another (loop 2) in the other direction. Because of
the antiparallel arrangement of the subunits in the dimer, loops 1 and 3 from
one subunit will come close to loop 2 from the other. The receptor-binding
epitope is built up by epitopes from all three loops, but the major contribu-
tion comes from loops 1 and 3 (Andersson et al., 1995; Schilling et al., 1998).
Despite the fact that there is no amino acid sequence similarity among PDGF,
nerve growth factor, and transforming growth factor-␤ (TGF-␤), all factors
have similar three-dimensional structures with cystine knots and dimeric
configurations (Murray-Rust et al., 1993).
B. Cell Types Producing PDGF
Several different cell types express PDGF, most often both the A and the B
polypeptide chains (Heldin and Westermark, 1999). However, less is known
about which cell types synthesize the newly discovered C chain. The A and
B chains are independently regulated, and the synthesis is often induced in
response to external stimuli. Thus, the production of PDGF by endothelial
Involvement of PDGF in Disease 3

cells is stimulated by thrombin (Daniel et al., 1986; Harlan et al., 1986), the
production by vascular smooth muscle cells is induced by mechanical strain
(Ma et al., 1999), and in several different cell types the production is induced
by low oxygen (Kourembanas et al., 1997) and by various growth factors and
cytokines. The PDGF A chain is also induced in uterus smooth muscle cells
during the physiological hypertrophy of pregnancy (Mendoza et al., 1990).
C. Processing and Compartmentalization of PDGF
The members of the PDGF family are synthesized as precursor molecules,
which need to undergo proteolytic processing before they can act on their
target cells. Thus, the A and B chain precursors are cleaved in their N ter-
mini, and the B chain in the C terminus, during their secretion from the pro-
ducer cells (Östman et al., 1992). The C chain contains an N-terminally lo-
cated CUB domain, which needs to be removed before the C chain can bind
to receptors (Li et al., 2000).
The A chain of PDGF occurs as two different splice forms; the long form,
but not the short form, contains a C-terminal basic sequence that mediates
interactions with negatively changed glycosaminoglycans in the extracellu-
lar matrix, as well as with structures inside the cell (Heldin and Westermark,
1999). Also, the B chain precursor contains a similar sequence. This means
that the long form of the A chain and the B chain is retained close to the pro-
ducer cell, whereas the short A chain may be more free to diffuse and to stim-
ulate cells at a distance.
D. PDGF Receptors
PDGF isoforms bind to structurally similar ␣- and ␤-tyrosine kinase re-
ceptors. The receptors have ﬁve immunoglobulin (Ig)-like domains in their
extracellular parts and tyrosine kinase domains intracellularly, which have
characteristic inserted sequences without homology to other kinases (re-
viewed in Heldin et al., 1998). The PDGF ␣ receptor gene is localized on
chromosome 4q12 close to the genes for the structurally related stem cell fac-
tor receptor, whereas the ␤ receptor gene is located on chromosome 5 close
to the gene for the structurally related colony stimulatory factor-1 receptor.
This suggests that this gene family has arisen through gene duplication.
E. Target Cells for PDGF
The classical target cells for PDGF, ﬁbroblasts and smooth muscle cells,
have both ␣ and ␤ receptors, albeit generally more ␤ receptors (reviewed in

Heldin and Westermark, 1999). Other cell types also express both receptors,
including kidney mesangial cells, Leydig cells, and certain cells in the central
nervous system, e.g., neurons, Schwann cells, and retinal pigment epithelial
cells. There are, however, several examples of cells that express only one of
the two receptor types. Thus, liver sinusoidal endothelial cells, astrocytes,
platelets, and megakaryocytes have only ␣ receptors, and Itoh cells of the liv-
er, myoblasts, capillary endothelial cells, pericytes, mammary epithelial cells,
T cells, myeloid hematopoietic cells and macrophages have only ␤ receptors.
F. Cellular Effects of PDGF Isoforms
The PDGF ␣- and ␤-receptor homo- and heterodimers induce similar, but
not identical, cellular effects. All dimeric combinations transduce mitogenic
signals. In some cell types, however, there are differences between the various
receptor dimers in their abilities to mediate actin reorganization. Whereas all
receptor dimers stimulate edge ruffling and loss of stress fibers, only ␣␤ and
␤␤ receptor dimers induce the formation of circular actin structures on the
dorsal surface of the cell (Eriksson et al., 1992). Also with regard to chemo-
taxis there is a difference between the various receptor dimers, at least in cer-
tain cell types; whereas ␤␤ and ␣␤ receptor dimers stimulate chemotaxis, ␣␣
receptor dimers inhibit the chemotaxis of smooth muscle cells and fibroblasts
(Koyama et al., 1996; Siegbahn et al., 1990). Both the ␣ and the ␤ receptor
mediate an increase in the intracellular Ca2⫹
concentration, but the ␤ recep-
tor is more efficient; in fact, the ␤ receptor-induced Ca2⫹
increase is depressed
by prior treatment of the cells with PDGF AA (Diliberto et al., 1992). PDGF
also exerts an antiapoptotic effect (Yao and Cooper, 1995), inhibits gap junc-
tional communication between cells (Hossain et al., 1998), and stimulates the
contraction of collagen gels (Clark et al., 1989; Gullberg et al., 1990).
III. INTRACELLULAR SIGNAL TRANSDUCTION
Ligand-induced dimerization of PDGF receptors activates a number of in-
tracellular signaling pathways, which ultimately lead to cell growth, changes
in cell morphology, and prevention of apoptosis. An extensive cross talk be-
tween different signaling pathways and the occurrence of parallel positive
and negative signals fine-tune and modulate the responses.
A. Receptor Activation
Because PDGF isoforms are dimeric molecules, their binding to receptors
causes receptor dimerization. The A and C chains of PDGF bind ␣ receptors,

whereas the B chain binds both ␣ and ␤ receptors with high affinity (Heldin
and Westermark, 1999; Li et al., 2000). Thus, PDGF AA and PDGF CC in-
duce ␣␣ receptor dimers, PDGF AB ␣␣ or ␣␤ receptor dimers, and PDGF BB
all three possible types of dimers (Fig. 1).
Whereas ligand binding to the outermost three Ig-like domains induces
dimerization of PDGF receptors (Heidaran et al., 1990), the dimeric com-
plex is further stabilized by direct interactions involving Ig-like domain 4
(Miyazawa et al., 1998; Omura et al., 1997; Shulman et al., 1997). Dimer-
ization of the receptor molecules brings the intracellular parts close to each
other, thereby allowing autophosphorylation in trans between the two re-
ceptors. The autophosphorylation occurs on specific tyrosine residues and
has two important consequences: phosphorylation of a conserved tyrosine
residue located in the activation loop of the kinase domain leads to an in-
crease of the kinase activity of the receptor and phosphorylation of several
tyrosine residues outside the kinase domain produces docking sites for sig-
naling molecules with SH2 domains (reviewed in Heldin et al., 1998).
B. Signaling via SH2 Domain Proteins
SH2 domains contain about 100 amino acid residues folded in such a way
that they recognize phosphorylated tyrosine residues in specific environments
Fig. 1 Several PDGF isoforms bind to two related receptors. The specificity in assembly of dif-
ferent dimeric receptor complexes of known PDGF isoforms is shown. Ig domains in the ex-
tracellular part of receptors are illustrated by circles, and split kinase domains in the intracel-
lular part are illustrated by rectangles.

(Pawson and Scott, 1997). A large number of SH2 domain-containing sig-
naling molecules have been shown to bind to autophosphorylated ␣ and/or
␤ receptors (reviewed in Heldin et al., 1998). Some of these molecules are
themselves enzymes, e.g., phosphatidylinositol-3⬘-kinase (PI-3-kinase) and
phospholipase C-␥ (PLC-␥), which both have the membrane phospholipid
phosphatidyl-inositol-4,5-bisphosphate (PtdIns-4,5-P2) as substrate, the Src
family of tyrosine kinases, the tyrosine phosphatase SHP-2, and the GTPase-
activating protein (GAP) for Ras. Other SH2 domains molecules, including
Grb2, Grb7, Crk, Nck, and Shc, lack enzymatic activity and serve as adap-
tors linking the receptors with downstream effector molecules. Also, Stat
molecules bind to PDGF receptors; after phosphorylation and activation
they are translocated to the nucleus where they act as transcription factors
and regulate the activity of specific genes.
In some cases, binding of SH2 domain proteins to the autophosphorylat-
ed receptors leads to their activation by phosphorylation on tyrosine residues
[e.g., PLC-␥ (Wahl et al., 1990) and Src (Erpel and Courtneidge, 1995)]. In
other cases, the binding causes a conformational change in the molecule,
which increases an intrinsic enzymatic activity [e.g., PI3-kinase (Backer et
al., 1992; Panayotou et al., 1992) and SHP-2 (Pluskey et al., 1995)], where-
as in still others, the signaling molecules may be constitutively active but their
binding to the receptors brings them to the localization in the cell where they
can interact with downstream effector molecules (e.g., the Grb2-Sos com-
plex and RasGAP).
Binding of each one of the SH2 domain-containing signaling molecules ini-
tiates a signal transduction pathway. This often involves activation of ki-
nases. A systematic analysis of proteins after PDGF stimulation of cells re-
vealed that over 100 changed their phosphorylation on serine/threonine or
tyrosine residues (Soskic et al., 1999). However, the notion that intracellu-
lar signal transduction consists of a number of linear signaling pathways that
mediate the different cellular effects of PDGF is an oversimplification.
Rather, there is an extensive cross talk between different signaling pathways,
creating an intracellular signaling network. Studies using microarrays to
monitor mRNA expression profiles support this notion; rather small differ-
ences were observed when signaling via wild-type receptors were compared
with signaling via tyrosine-mutated receptors (Fambrough et al., 1999).
Irrespective of the extensive cross talk between signaling pathways, PI3-
kinase has been shown to be particularly important for cell motility and anti-
apoptotic responses. Thus, PI3-kinase, which produces PtdIns-3,4,5-P3, is
essential for PDGF-induced actin reorganization and chemotaxis (Wenn-
ström et al., 1994a,b). Important downstream components that mediate
these effects are members of the Rho family of small GTPases (Hawkins et
al., 1995; Hooshmand-Rad et al., 1997). Interestingly, in addition to an ear-
ly wave of PI3-kinase activity, important for morphological changes, PDGF
also induces a second wave of PI3-kinase activity with a maximum after 4–

6 hr which is important for the initiation of DNA synthesis (Jones et al.,
1999). The antiapoptotic effect of PI3-kinase involves activation of the ser-
ine/threonine kinase Akt (Dudek et al., 1997; Kauffmann-Zeh et al., 1997)
and NF-␬B (Romashkova and Makarov, 1999).
PLC-␥, which cleaves PtdIns-4,5-P2 to diacylglycerol and Ins-1,4,5-P3, ap-
pears not to be essential for any of the PDGF responses. However, in cells
deprived of other signals, PLC-␥ can mediate the stimulation of cell growth
(Valius and Kazlauskas, 1993); upon overexpression, PLC-␥ makes cells
more susceptible to migratory responses (Rönnstrand et al., 1999). It has
been suggested that calcium mobilization downstream of PLC-␥ is required
for the stimulation of sphingosine kinase by PDGF, which has been linked to
growth stimulation in certain cell types (Olivera et al., 1999).
Activation of Ras, which leads to activation of the MAP kinase cascade,
is important for the mitogenic effect of PDGF. Ras is activated by the bind-
ing to the PDGF receptor, directly or indirectly, of the adaptor molecule Grb2
in complex with the nucleotide exchange molecule Sos1 (Bos, 1997). Sos1
converts Ras from its inactive GDP-bound form to the active GTP-bound
form. Also the tyrosine kinase Src has been shown to be important for the
mitogenic effect of PDGF (Twamley-Stein et al., 1993). In addition, Src may
affect cytoskeletal organization and cell morphology via activation of an-
other tyrosine kinase, Abl (Plattner et al., 1999).
C. Modulation of Signaling
There are several examples of inhibitory signals induced at the PDGF re-
ceptor in parallel to the stimulatory signals and which modulate the effects
(reviewed in Heldin, 1997). Thus, at the same time as the Grb2/Sos1 com-
plex binds to the PDGF ␤ receptor and activates Ras, another molecule, Ras-
GAP, also binds to the receptor and counteracts Ras activation by convert-
ing active Ras-GTP to inactive Ras-GDP (Heidaran et al., 1993). Moreover,
the tyrosine phosphatase SHP-2, through its ability to dephosphorylate the
autophosphorylated receptor or its substrates, may also modulate signaling
via PDGF receptors (Klinghoffer and Kazlauskas, 1995). Another example
is a Src-like adaptor protein (Slap), which interacts with juxtamembrane au-
tophosphorylation sites and inhibits PDGF signaling (Roche et al., 1998).
One mechanism whereby the balance between stimulatory and inhibitory
signals may be altered is through differential autophosphorylation of the re-
ceptor. One such example has been unraveled. The ␤ receptor in a homo-
dimeric complex is efﬁciently autophosphorylated on Tyr771, which binds
RasGAP. In contrast, this tyrosine residue is not autophosphorylated when
the ␤ receptor forms a heterodimer with the ␣ receptor, which itself does not
bind RasGAP (Ekman et al., 1999). Thus, PDGF AB, which preferentially

forms ␣␤ heterodimeric receptors, activates Ras more efficiently than PDGF
BB, which forms homodimeric as well as heterodimeric receptor complexes.
This may explain why PDGF AB gives a stronger mitogenic effect than PDGF
BB on cells having both ␣ and ␤ receptors.
It is possible that signaling via PDGF receptors can be modulated by the
selective dephosphorylation of specific autophosphorylation sites. Thus, the
tyrosine phosphatase SHP-2 has been shown to selectively dephosphorylate
Tyr771 in a process that is activated by binding of the cell to fibronectin (De-
Mali et al., 1999). Consistent with this finding, a SHP-2-dependent increased
activation of MAP kinase has been observed (Zhao and Zhao, 1999), which
may result from dephosphorylation of Tyr771 followed by a decreased bind-
ing of GAP and increased activation of Ras. Also the tyrosine phosphatase
DEP-1 has been shown to selectively dephosphorylate certain autophospho-
rylated tyrosine residues in the PDGF ␤ receptor (Kovalenko et al., 2000).
IV. IN VIVO FUNCTION OF PDGF
PDGF has important roles in the regulation of cell growth, differentiation,
and migration during the embryonal development, as well as during wound
healing. PDGFs also have specialized functions in the vascular system as well
as in the homeostasis of the connective tissue.
A. Embryonic Development
Studies on the expression of PDGF and PDGF receptors by different cells
during the development have revealed that PDGFs are often produced by cer-
tain epithelial cells and PDGF receptors by the adjacent mesenchymal cells,
suggesting a paracrine mode of action (reviewed in Heldin and Westermark,
1999). Moreover, the expression patterns of the receptors suggest that PDGF
have important roles in the development of connective tissue compartments
in different organs. Interestingly, the different PDGF isoforms show differ-
ent expression patterns. Also, PDGF A and C chains, which both bind to the
␣ receptor, are produced by completely different subsets of epithelial cells,
e.g., in the embryonal kidney (Li et al., 2000).
The notion that PDGFs are important for the development of connective
tissue compartments is supported by experiments in which the genes for
PDGF A or B chains, or receptors, were knocked out in the mouse. Target-
ing of the B chain or the ␤ receptor led to severe effects on kidney develop-
ment with a total absence of mesenchymal cells, leading to a poor filtration
in the glomeruli (Levéen et al., 1994; Soriano, 1994). Also, blood vessels

were affected with a dilated aorta and characteristic bleeding at about the
time of birth, which led to the death of the animals at that time. This is like-
ly to be due to a deficiency of newly formed blood vessels to attract pericytes
(Lindahl et al., 1997a) and in the development of the vascular smooth mus-
cle cell layer (Hellström et al., 1999). In order to circumvent the embryonic
lethality of PDGF ␤-receptor-deficient animals, a chimeric analysis was un-
dertaken, which revealed that, during the development, the ␤ receptor is im-
portant for all muscle lineages, but not for fibroblast and endothelial lineages
(Crosby et al., 1998). However, during wound healing, expression of ␤ re-
ceptors on fibroblasts and endothelial cells is essential for efficient connec-
tive tissue repair (Crosby et al., 1999).
PDGF A-chain knockout mice showed defects in the development of the
alveoli of the lungs, giving an emphysema-like phenotype, which led to death
of the mice at about 3 weeks of age (Boström et al., 1996). The defect is due
to defect spreading of alveolar smooth muscle cell progenitors, whereby the
alveolar walls do not develop (Lindahl et al., 1997b). PDGF A also stimu-
lates the proliferation of different types of dermal mesenchymal cells and is,
together with sonic hedgehog, involved in the development of hair follicles
(Karlsson et al., 1999).
Knockout of the ␣ receptor gave a more severe phenotype compared to the
A chain knockout, which is expected, as this receptor binds PDGF A and B
chains, as well as C chains. The phenotype includes cranial malformations
and a deficiency of myotome formation (Soriano, 1997). The ␣ receptor is
affected in the spontaneous Patch mouse mutant, which has a phenotype
similar to the ␣ receptor knockout mouse, but in addition has a coat color
defect that possibly is due to perturbed expression of the neighboring stem
cell receptor gene (Stephenson et al., 1991).
There is also expression of PDGF receptors on cells of nonmesenchymal
origin, e.g., in the central nervous system, indicating that PDGF has func-
tional importance, not only for connective tissue cells. The PDGF ␣ recep-
tor is expressed in the neuronal tube at embryonal day (ED)9 in the mouse
and occurs in the brain, brain stem, and spinal cord on E13.5 (Orr-Urtreger
et al., 1992; Schatteman et al., 1992). At later stages, after E16, the ␣ re-
ceptor is expressed by oligodendroglial–astroglial precursor cells (O-2A
cells) (Pringle et al., 1992), and PDGF has been shown to have an important
role in the growth and differentiation of this cell type (Fruttiger et al., 1999).
In culture, O-2A cells undergo premature differentiation in the absence of
growth factors; PDGF, in combination with basic fibroblast growth factor,
completely blocks differentiation of O-2A cells and causes functional im-
mortalization of the cells (Wolswijk and Noble, 1992).
Through its effect on oligodendrocyte development, PDGF has an impor-
tant role in the myelination of cells (Butt et al., 1997; Fruttiger et al., 1999;
Redwine and Armstrong, 1998). Thus, transplantation of PDGF-producing

cells was found to lead to an expansion of the oligodendrocyte compartment
(Barres et al., 1992), and PDGF was shown to reduce chemically induced rat
oligodendrocyte death and enhance myelination in vitro (Fressinaud et al.,
1996). Also the myelinating cells in the periferal nervous system, Schwann
cells, respond to PDGF through their expression of ␤ receptors (Davis and
Stroobant, 1990; Eccleston et al., 1990). These observations suggest an im-
portant therapeutic possibility for PDGF, i.e., to induce remyelination.
PDGF receptors are also expressed on certain neurons (Oumesmar et al.,
1997; Smits et al., 1991), and PDGF has been shown to have neuroprotec-
tive and neurotrophic effects on cultured rat dopaminergic neurons (Othberg
et al., 1995; Pietz et al., 1996). Consistent with a role in neuroprotection and
regeneration, PDGF and PDGF receptors were shown to be upregulated in
infarcted human brain tissue and in facial nuclei of the rat after axotomi
(Hermanson et al., 1995). Moreover, PDGF and PDGF receptor expression
is increased after ischemic stroke (Krupinski et al., 1997), and administra-
tion of PDGF BB was found to protect the rat brain after experimental focal
ischemia (Sakata et al., 1998).
B. Wound Healing
In the adult, PDGF has been shown to stimulate wound healing. PDGF
does not appear to alter the normal sequence of repair, but increases its rate.
Local application of PDGF leads to an increased formation of granulation
tissue (Grotendorst et al., 1985; Sprugel et al., 1987) and faster wound heal-
ing in rat skin (Pierce et al., 1988). PDGF also stimulates wound healing in
other animal models, e.g., excisional wounds in rabbit ear (Mustoe et al.,
1991) and burn injuries in pigs (Danilenko et al., 1995). In addition to local
application of PDGF, beneficial effects on wound healing have also been ob-
served after particle-mediated delivery of PDGF cDNAs (Eming et al., 1999)
or after adenovirus-mediated overexpression of PDGF (Liechty et al., 1999)
in the wounded area. PDGF was shown to stimulate wound healing in pa-
tients (Robson et al., 1992), and topical application of recombinant PDGF
BB (becaplermin) has been subject to clinical trials and shown to have ben-
eficial effects on the healing of different types of wounds (reviewed by
LeGrand, 1998).
The healing of soft tissues involves several steps, including reepitelializa-
tion, angiogenesis, and extracellular matrix deposition. PDGF acts on sev-
eral cell types involved in the healing process. It stimulates mitogenicity
and chemotaxis of fibroblasts and smooth muscle cells, and chemotaxis of
neutrophils and macrophages. PDGF also stimulates the production of var-
ious matrix molecules, including fibronectin (Blatti et al., 1988), collagen
(Canalis, 1981), proteoglycans (Schönherr et al., 1991), and hyaluronic acid

(Heldin et al., 1989). In addition, PDGF may indirectly promote wound heal-
ing, e.g., by stimulating macrophages to release other growth factors and cy-
tokines that affect the healing process.
PDGF may also have a role at later stages of wound healing. It stimulates
contraction of collagen gels in vitro (Clark et al., 1989; Gullberg et al., 1990),
suggesting that it may affect wound contraction in vivo. Moreover, PDGF
stimulates the production and secretion of collagenase by fibroblasts (Bauer
et al., 1985), suggesting a role in the remodeling phase of wound healing.
C. Vascular System
PDGF ␤ receptors are expressed by capillary endothelial cells, and PDGF
has been shown to have an angiogenic effect (Battegay et al., 1994; Edelberg
et al., 1998; Nicosia et al., 1994; Risau et al., 1992; Sato et al., 1993). The
effect is, however, weaker than that of bona fide angiogenic factors of the
VEGF or FGF families. PDGF is not likely to be important for the initial for-
mation of blood vessels, vasculogenesis, as no apparent vascular abnormal-
ity was observed in PDGF or PDGF receptor knockout mice. However, as
mentioned earlier, PDGF may have a significant role at later stages in the for-
mation of blood vessels, through its ability to recruit pericytes and stimulate
the development of vascular smooth muscle cells, which have important
functions in reinforcing the structural integrity of the vessels (Hellström et
al., 1999; Lindahl et al., 1997a).
PDGF may also have a role in the regulation of the tonus of blood vessels.
PDGF BB has been shown to stimulate endothelial cells to release NO and
thereby relax rat aorta (Cunningham et al., 1992) and to lower the blood
pressure through increased vascular compliance. However, PDGF has also
been reported to induce the constriction of different types of blood vessels
(Berk et al., 1986; Sachinidis et al., 1990).
Another potentially important function of PDGF in the vascular system is
to exert a feedback control on platelet aggregation. Human platelets, which
are a rich source of PDGF, have PDGF ␣ receptors (Vassbotn et al., 1994a).
After thrombin-induced platelet aggregation, PDGF and other constituents
of the ␣-granulae are released. PDGF then binds to ␣ receptors on the
platelets, thereby inhibiting platelet aggregation through an autocrine feed-
back control function (Bryckaert et al., 1989; Vassbotn et al., 1994a).
D. Tissue Homeostasis
In tissues, macromolecules are exchanged between the vessels and the ex-
tracellular compartment. The exchange is regulated by the interstitial fluid

pressure, which normally is slightly negative. PDGF has been shown to have
an important role in the control of the interstitial fluid pressure, probably
through its ability to stimulate interactions between connective tissue cells
and extracellular matrix molecules (Rodt et al., 1996). The PI3-kinase path-
way is important for this effect, as mice with the PDGF ␤ receptor replaced
with a ␤ receptor mutant lacking the two tyrosine residues involved in
PI3-kinase activation were unable to control the interstitial fluid pressure
(Heuchel et al., 1999).
V. PDGF IN DISEASE
Whereas PDGF has important roles during embryonal development and
wound healing, as described earlier, overactivity of PDGF has been impli-
cated in the pathogenesis of a number of serious diseases, including cancer,
as well as other disorders characterized by excessive cell growth, such as ath-
erosclerosis and various fibrotic conditions.
A. Cancer
1. AUTOCRINE PDGF STIMULATION IN TUMORS
Because glial cells, fibroblasts, and smooth muscle cells are normal target
cells of PDGF, tumors from these cell types have been analyzed for autocrine
PDGF stimulation. In glioblastomas, a large fraction of analyzed tumors
demonstrated coexpression of PDGF ␣ receptor and PDGF A or B chains
(Fleming et al., 1992; Guha et al., 1995; Hermanson et al., 1992). The no-
tion that PDGF autocrine loops play a causative role in the development of
glioblastomas is further supported by the observations that intracranial in-
jections with SSV, carrying a viral form of the PDGF B chain, into marmo-
sets, or a recombinant PDGF B-chain encoding retrovirus into mice, cause
glioblastoma (Deinhardt, 1980; Uhrbom et al., 1998).
Soft tissue sarcomas have also been suggested to contain PDGF autocrine
loops (Smits et al., 1992; Wang et al., 1994). Coexpression of ligand and re-
ceptor was observed in clinical samples of fibroblast-derived tumors such as
dermatofibroma and malignant fibrous histiocytoma. The fibrosarcoma-
inducing potential of PDGF autocrine loops has also been demonstrated
through tumor development on infections with PDGF B chain encoding
viruses (Deinhardt, 1980; Pech et al., 1989). Dermatofibrosarcoma protu-
berans (DFSP) and the juvenile form giant cell fibroblastoma are skin tumors
of intermediate malignancy. In most, if not all, of these tumors, a transloca-

tion that fuses the collagen 1A1 gene to the PDGF B chain gene occurs
(O’Brien et al., 1998; Simon et al., 1997). Transfection of collagen 1A1/
PDGF B fusion genes into NIH/3T3 cells has confirmed their transforming
potential (Greco et al., 1998; Shimizu et al., 1999). Biochemical studies have
also demonstrated that fusion proteins are processed to mature PDGF BB
(Shimizu et al., 1999). Taken together, these observations suggest that au-
tocrine PDGF stimulation is important in the development of DFSP.
Coexpression of PDGF and PDGF receptors, consistent with autocrine
growth stimulation, has also been observed in various other types of human
tumors by immunohistochemical staining and by in situ analyses of mRNA
expression. Examples include meningiomas (Black et al., 1994; Figarella-
Branger et al., 1994; Todo et al., 1996) ependymomas (Black et al., 1996),
pituitary adenomas (Leon et al., 1994), mesotheliomas (Langerak et al.,
1996), melanomas (Barnhill et al., 1996), choriocarcinomas (Holmgren et
al., 1993), ovarian cancer (Henriksen et al., 1993), neuroendocrine tumors
(Chaudhry et al., 1992), prostatic cancer (Fudge et al., 1996), pancreatic can-
cer (Ebert et al., 1995), gastric cancer (Chung and Antoniades, 1992), and
lung cancer (Antoniades et al., 1992). Except for the gene translocation
in DFSP, the mechanisms behind dysregulated PDGF expressions are not
known. More recently, PDGF ␤ receptor antibodies specifically recognizing
the autophosphorylated receptor were used to demonstrate activated PDGF
␤ receptors in human meningioma (Shamah et al., 1997).
The ligand:receptor interaction in cells with autocrine PDGF BB stimula-
tion has been analyzed extensively. It has been established that PDGF binds
to its receptor already in the endoplasmatic reticulum (Fleming et al., 1989;
Keating and Williams, 1988); however, for an efficient mitogenic signal to
appear, the ligand:receptor complex needs to be translocated to the plasma
membrane, possibly because important downstream signaling components
are located there (Fleming et al., 1989; Hannink and Donoghue, 1988; Val-
geirsdóttir et al., 1995).
2. LIGAND-INDEPENDENT ACTIVATION OF PDGF
RECEPTORS IN TUMORS
Whereas the mechanism for dysregulated expression of PDGF receptors in
tumors in most cases is unknown, in some cases, overexpression of the PDGF
␣ receptor in glioblastoma is associated with amplification of the PDGF ␣
receptor gene (Fleming et al., 1992; Hermanson et al., 1996). Amplification
occurred in a subset of tumors also characterized by loss of heterozygosity
on chromosome 17p (Hermanson et al., 1996).
Chronic myelomonocytic leukemia (CMML) is a rare disease associated
with the activation of PDGF ␤ receptors through a translocation that gives
rise to a ligand-independent constitutively active form of the PDGF ␤ recep-

tor (Carroll et al., 1996; Golub et al., 1994; Jousset et al., 1997). In CMML,
the PDGF receptor is activated by dimerization mediated by the fusion part-
ner, the Ets-like transcription factor TEL (Carroll et al., 1996; Jousset et al.,
1997; Sjöblom et al., 1999). The disease-causing potential of the TEL/PDGF
␤ receptor fusion gene has been demonstrated in two transgenic mouse mod-
els using immunoglobulin and CD11A promotors (Ritchie et al., 1999;
Thomasson et al., 1999).
The transforming potential of the PDGF receptor pathway has also been
highlighted by studies of the E5 oncoprotein of bovine papilloma virus type
1, which acts by activation of the PDGF ␤ receptor (reviewed in DiMaio
et al., 1998). The mechanism involve E5-mediated oligomerization of the
PDGF ␤ receptor, whereby the receptor is also activated in the absence of
ligand.
3. PARACRINE PDGF EFFECTS ON TUMOR STROMA, TUMOR
ANGIOGENESIS, AND METASTASIS
In a solid tumor, cells of the stromal, vessel, and tumor compartments in-
teract in an interdependent way, which is crucial for the development and
progression of tumors, as well as for tumor angiogenesis. One demonstra-
tion of the importance of the stromal compartment was the identification of
stromal fibroblasts as the major source of VEGF in mammary tumors de-
veloping in transgenic mice expressing the polyoma middle T antigen under
the control of the MMTV promotor (Fukumura et al., 1998).
The potential of paracrine PDGF stimulation of stromal fibroblasts in tu-
mor growth has been demonstrated by the forced overexpression of the
PDGF B chain in two cell types lacking PDGF receptor expression, WM9
melanoma cells (Forsberg et al., 1993), and HaCaT keratinocytes (Skobe and
Fusenig, 1998). PDGF-expressing WM9 cells formed tumors that grew more
rapidly, had less necrosis, and had more well-vascularized connective tissue
septa as compared to parental cells. In the case of HaCaT cells, PDGF B ex-
pression led to conversion of cells that formed benign tumors in mice through
mechanisms proposed to involve both direct effects on stroma formation and
angiogenesis and indirect effects on the keratinocytes mediated by activated
stromal cells.
Many common solid tumors, including colorectal adenocarcinoma, lung
carcinomas, and breast carcinomas, display PDGF producing tumor cells
and PDGF ␤ receptor expressing stromal fibroblasts (Bhardwaj et al., 1996;
Kawai et al., 1997; Lindmark et al., 1993; Sundberg et al., 1997; Vignaud
et al., 1994). In addition to mediating growth promoting effects on tumor
stroma, PDGF receptors expressed on stromal fibroblasts and pericytes may
also be involved in the regulation of tumor interstitial fluid pressure and
transcapillary transport (Pietras et al., 2000).

Antiangiogenic therapy has appeared as an attractive novel way of inter-
fering with tumor growth (reviewed by Ferrara and Alitalo, 1999). Numer-
ous studies have demonstrated striking effects in animal models of tumor
growth by targeting angiogenic growth factors such as VEGF, FGF, and an-
giopoietin-1 (Ferrara and Alitalo, 1999). Angiogenic activity of PDGF BB
has been demonstrated in rings of rat aorta, in the chick chorioallantoic
membrane assay, and in cocultures of microvascular fragments and myofi-
broblasts (Battegay et al., 1994; Nicosia et al., 1994; Risau et al., 1992; Sato
et al., 1993). Furthermore, PDGF BB might be of particular importance for
pericyte recruitment, required for structural and functional integrity of cap-
illaries, as determined by the phenotype of PDGF B chain knockout mice
(Crosby et al., 1998; Lindahl et al., 1997a). PDGF ␤ receptor expression is
upregulated in endothelial cells in glioblastomas (Hermanson et al., 1988;
Plate et al., 1992), and high expression has also been demonstrated in peri-
cytes of the microvasculature of colon cancers (Sundberg et al., 1993). PDGF
receptor signaling might thus contribute by various mechanisms to tumor
angiogenesis, i.e., indirectly through action on stromal cells responsible for
the production of VEGF or by direct action on pericytes or endothelial cells.
Matrix metalloproteinases are extracellular matrix degrading enzymes
contributing to the metastatic process. PDGFs induce the production of
MMP-1 and -2 in cultured fibroblasts and embryonic bronchial arch ex-
plants (Alvares et al., 1995; Robbins et al., 1999). Induction of MMP-9 by
PDGF BB has been demonstrated in vascular smooth muscle cells (Fabunmi
et al., 1996). Furthermore, a critical role for PDGF ␣ receptor signaling in
the physiological control of MMP-2 production is indicated by the reduced
MMP-2 expression in the bronchial arch and heart tissue of mice homo-
zygous for the Patch mutation, which inactivates the PDGF ␣ receptor (Rob-
bins et al., 1999). It can thus be hypothesized that PDGF signaling contrib-
utes to metastasis and invasion by MMP induction. Experimental support
for a prometastatic function of PDGF ␣ receptor signaling was provided with
the observation that overexpression of the full-length PDGF ␣ receptor in
the 3LL clone of Lewis lung carcinoma cells promotes lung metastasis
(Fitzer-Attas et al., 1997). A positive correlation between PDGF A chain ex-
pression and lymph node metastasis was also noted when 32 primary inva-
sive breast tumors were examined (Anan et al., 1996). The availability of
specific PDGF antagonists (see later) will allow further studies on the role of
PDGF stimulation in invasion and metastasis.
B. Atherosclerosis
Atherosclerosis, as well as premature vessel obstruction following percu-
taneous transluminal angioplasty, bypass grafting, or transplantations, is as-

sociated with smooth muscle cell proliferation and migration and subsequent
matrix deposition. The potency of PDGF as a smooth muscle cell mitogen
and chemotactic agent has prompted studies exploring the possibility that
PDGF receptor signaling contributes to these pathological processes.
Immunohistochemical analysis of human atherosclerotic lesions has dem-
onstrated a disease-associated upregulation of PDGF AB and BB production,
as well as PDGF ␤ receptor expression, compatible with a causative role for
PDGF in the development of atherosclerosis (Ross et al., 1990; Rubin et al.,
1988). The ␤ receptor appears to be more important than the ␣ receptor in
mediating the atherosclerotic reaction (Giese et al., 1999).
A role of PDGF receptor signaling in the more acute process of neointima
formation following percutaneous transluminal coronary angioplasty is sug-
gested by immunohistochemical and in situ mRNA expression studies of hu-
man coronary arteries (Tanizawa et al., 1996; Ueda et al., 1996). Increased
PDGF ␤ receptor expression was observed in smooth muscle cells in areas
undergoing repair; in these areas, PDGF AB/BB production was found in
macrophages, spindle cells, endothelial cells, and smooth muscle cells. In-
creased expression of PDGF ligands and receptors has also been demon-
strated in a number of experimental injury models of restenosis, as well as
in the neointima forming in polytetraﬂuoroethylene vascular grafts implant-
ed in baboons (Golden, 1991; Kanzaki et al., 1994; Kraiss et al., 1993; Ma-
jesky et al., 1990; Uchida et al., 1996).
Concerning transplantation-induced vasculopathies, no report yet exists
on the situation in human transplanted tissues. However, the neointima of
rat aortic and cardiac allografts has been analyzed, and the pattern of PDGF
and PDGF receptor expression suggests an involvement of PDGF-induced
smooth muscle cell migration and proliferation in this type of neointima for-
mation (Akyürek et al., 1996; Lemström and Koskinen, 1997).
The mechanism underlying the induction of PDGF ligand and receptor ex-
pression in atherosclerotic conditions remains incompletely understood.
Rheological factors, such as ﬂow rate and shear stress, might contribute to
increased PDGF expression. In rabbit and baboon models, where shear stress
was manipulated experimentally, a reduction in shear stress was associated
with increased PDGF ligand production by endothelial cells and concomi-
tant increased receptor expression in smooth muscle cells (Kraiss et al., 1996;
Mondy et al., 1997).
The possibility that PDGF stimulation plays a causative role in the forma-
tion of neointima has been analyzed by studying the effects of infusion of
PDGF BB in the rat carotid injury model of restenosis and of liposome-
mediated transfer of the PDGF B chain gene into porcine arteries. In the rat
model, a large increase in intimal thickening was observed, despite only mod-
est effects on smooth muscle cell proliferation, suggesting predominantly
promigratory effects of PDGF BB (Jawien et al., 1992). Intimal thickening

was also observed in the porcine model after PDGF B chain gene transfer,
and further analysis of lesions indicated stimulatory effects of PDGF BB on
smooth muscle cell proliferation and migration, as well as extracellular ma-
trix deposition (Nabel et al., 1993; Pompili et al., 1995).
C. Lung Fibrosis
Lung fibrosis is associated with fibroblast proliferation and accumulation
of extracellular matrix, processes known to be stimulated by PDGF. A role
for PDGF in various forms of lung fibrosis is suggested by observations of
PDGF overexpression in patients affected by these diseases, as well as by var-
ious lung fibrosis model studies where PDGF induction has been demon-
strated and where, more recently, effects of PDGF antagonists have been ob-
served.
Studies on patient samples have shown that increased PDGF B chain pro-
duction by invading macrophages, as well as by epithelial cells, occurs in as-
sociation with idiopathic pulmonary fibrosis and bronchiolitis obliterans-
organizing pneumonia as determined by in situ and immunohistochemical
analyses (Antoniades et al., 1990; Aubert et al., 1997; Vignaud et al., 1991).
Analysis of lavage fluids has also demonstrated increased PDGF levels in
lavage fluid from patients with acute diffuse lung injury (Snyder et al., 1991),
obliterative bronchiolitis after lung transplantation (Hertz et al., 1992), Her-
mansky–Pudlak syndrome (Harmon et al., 1994), histiocytosis X (Uebelho-
er et al., 1995), and coal workers pneumoconiosis (Vanhee et al., 1994).
Increased production of PDGF in animal models has also been shown
after the induction of lung fibrosis by hyperoxia (Powell et al., 1992), bleo-
mycin instillation (Walsh et al., 1993), or chrysotile asbestos (Liu et al.,
1997). Transgenic mice, expressing the B chain of PDGF in the lung under
the control of the lung surfactant protein promoter, showed abnormalities
in the developing and adult lung, involving both emphysema and fibrotic
lung disease (Hoyle et al., 1999). Conversely, decreased PDGF expression is
associated with the reduced fibrosis that occurs after pirfenidone treatment
in the bleomycin hamster model of lung fibrosis (Gurujeyalakshmi et al.,
1999). Furthermore, direct evidence for the fibrotic potential of PDGF in the
lung was obtained with the demonstration of pulmonary cell proliferation
and collagen deposition after the intratracheal injection of PDGF-BB (Yi et
al., 1996).
D. Glomerulonephritis
Evidence indicates that PDGF stimulation has a large impact on the patho-
genesis of renal disease. In 1989, PDGF receptor upregulation was demon-

strated in kidney specimens from rejected kidney transplants and in cases of
glomerulonephritis (Fellström et al., 1989). Subsequent studies have com-
pared PDGF receptor expression in human samples of proliferative and non-
proliferative glomerulonephritis and observed increased receptor expression
in proliferative forms (Gesualdo et al., 1994). Analysis of patient biopsies
has also revealed increased PDGF expression in cases of IgA glomerulo-
nephritis (Niemir et al., 1995) and renal vascular rejection (Alpers et al.,
1996). Increased PDGF A chain production is associated with congenital
multicystic renal dysplasia (Liapis et al., 1997). In agreement with these find-
ings, upregulation of PDGF ligand and receptors was observed in rat and
mouse models of proliferative glomerulonephritis (Gesualdo et al., 1991;
Iida et al., 1991; Yoshimura et al., 1991).
The approach of forced overexpression of PDGF or infusion of recombi-
nant PDGF has also been used to evaluate the role of PDGF in kidney fibro-
sis (Floege et al., 1993; Isaka et al., 1993; Tang et al., 1996). When the ef-
fects of PDGF B chain and TGF-␤ gene transfer were compared, it was noted
that both genes induced glomerulosclerosis; however, TGF-␤ stimulated pre-
dominantly extracellular matrix accumulation, whereas the PDGF B chain
induced a proliferative response (Isaka et al., 1993). Both glomerular cell
proliferation and tubulointerstitial hyperplasia have been observed after
PDGF BB infusion (Floege et al., 1993; Tang et al., 1996).
VI. PDGF AND PDGF RECEPTORS AS DRUG TARGETS
Considering the likely involvement of PDGF overactivity in several serious
disorders, clinically useful PDGF antagonists are highly warranted. Various
types of PDGF antagonists have been developed and have beneficial effects
in various animal models; their potential clinical utility is currently evaluat-
ed in patients.
A. PDGF Antagonists
Antagonists targeting each of the various steps in ligand-induced PDGF re-
ceptor activation, i.e., ligand binding, receptor dimerization, and subsequent
activation of the receptor kinase, have been developed (Fig. 2). There are ad-
vantages and disadvantages for each of these approaches. In general, tyro-
sine kinase receptors display much larger diversity in their extracellular do-
mains than in their kinase domains, thus suggesting that antagonists specific
for the PDGF receptors would be easier to obtain by targeting ligand:recep-
tor and receptor:receptor interactions than by targeting the kinase domain.
Another general advantage with compounds acting extracellularly is that

they are less likely to be sensitive to the multiple drug resistance phenotype,
which involves cellular excretion of compounds and is commonly observed
in solid tumors. A principal disadvantage with ligand:receptor and recep-
tor:receptor interactions as drug targets is that they often involve interac-
tions between large, ﬂat surfaces of proteins and are thus inherently more
difﬁcult to block with low molecular weight compounds than the kinase do-
main where the ATP-binding pocket provides a good target for small mole-
cules. Furthermore, interactions between PDGF and its receptors during au-
tocrine stimulation occur in the secretory pathway, suggesting that PDGF
autocrine stimulation is less sensitive to extracellularly acting agents com-
pared to situations where PDGF receptor stimulation occurs through
paracrine signaling.
In addition, antagonists acting through various genetic approaches, in-
cluding dominant-negative forms of ligand or receptor, as well as antisense-
Fig. 2 Mechanism of PDGF receptor activation and schematic illustration of different possi-
ble levels of action of PDGF antagonists. Thus, antagonists may suppress the expression of
PDGF or PDGF receptors, bind to PDGF or PDGF receptors in such a way that ligand:recep-
tor interaction is prevented, bind to the epitope in the receptor involved in receptor:receptor in-
teractions and thereby prevent receptor dimerization, or may inactivate the kinase.

mediated suppression of ligand or receptor expression, have been success-
fully used to block PDGF signaling (Fig. 2).
1. ANTAGONISTS INTERFERING WITH
LIGAND:RECEPTOR INTERACTIONS
Neutralizing polyclonal and monoclonal antibodies with various specifi-
cities against the PDGF isoforms have been raised in rabbits, goats, sheep,
and mice (Ferns et al., 1991; Rutherford et al., 1997b; Thyberg et al., 1990;
Vassbotn et al., 1990). Neutralizing mouse monoclonal antibodies specific
for the PDGF ␣ or ␤ receptor have also been generated (Koyama et al., 1994,
1996, 1998; LaRochelle et al., 1993; Lokker et al., 1997; Ramakrishnan et
al., 1993; Tiesman and Hart, 1993). Therapeutic effects of antibodies against
ligand or receptor have been obtained in animal models of restenosis and
glomerulonephritis (Ferns et al., 1991; Giese et al., 1999; Hart et al., 1999;
Johnson et al., 1992; Rutherford et al., 1997b).
SELEX aptamers are inhibitory molecules that have been raised against a
number of protein targets, including various growth factors (Floege et al.,
1999a). Their mechanism of action is similar to ligand-neutralizing anti-
bodies in that aptamers also bind to the target growth factor and interfere
with receptor binding. Three DNA aptamers against PDGF AB and BB, be-
tween 37 and 45 nucleotides in length, were isolated after a screen of an
oligonucleotide library (Green et al., 1996). All three aptamers neutralized
PDGF AB- and BB-induced DNA replication with IC50
values of 1–10 nM.
Photo-cross-linking of one of the aptamers, 20t, demonstrated interaction
between the aptamer and the loop III region of the PDGF B chain, implicat-
ed in receptor binding. More recently, a PEG-conjugated and chemically
modified form of the PDGF targeting aptamer 36t has been used successful-
ly to block glomerulonephritis (Floege et al., 1996b) and arterial restenosis
(Leppänen et al., 2000a) in rat models of these diseases.
A third type of antagonist targeting ligand:receptor interactions are solu-
ble PDGF receptors (Duan et al., 1991; Miyazawa et al., 1998; Rooney et
al., 1994). Such molecules have been shown to block PDGF with IC50 val-
ues of 10–100 nM in tissue culture experiments. Furthermore, Ig or GST fu-
sion variants of these proteins have been made (Heidaran et al., 1995; Lep-
pänen et al., 2000b); it appears that the ligand-independent dimerization
provided by the Fc and GST domains, respectively, yields proteins with sig-
nificantly improved IC50 values.
Linear or cyclic peptides containing sequences from loop 1 and loop 3 of
PDGF B chain have also been shown to antagonize PDGF, albeit at high con-
centrations (Brennand et al., 1997b; Engström et al., 1992). The evaluation
of these peptide antagonists is complicated by the fact that they also affect
cell viability; whereas linear peptides showed unspecific cytotoxicity, the

cyclic peptide was claimed to induce apoptosis (Brennand et al., 1997a,b;
Engström et al., 1992).
As revealed by the determination of the three-dimensional structures of
several ligand:receptor complexes, interactions between growth factors and
their cognate receptors, like most other protein:protein interactions, involve
protein contacts distributed over rather large surface areas (Starovasnik et
al., 1997). This has raised general doubts about the possibility to generate
low molecular weight PDGF antagonists targeting ligand:receptor interac-
tions. However, in a report from 1994, 2-bromomethyl-5-chlorobenzene sul-
fonylphtalimide was shown to block receptor binding of PDGF (Mullins et
al., 1994). The compound also showed in vivo effects in a rat model of
restenosis after oral administration.
2. ANTAGONISTS INTERFERING WITH
RECEPTOR DIMERIZATION
As illustrated in Fig. 2, the signaling PDGF:receptor complex is stabilized
not only by ligand:receptor interactions, but also by receptor:receptor inter-
actions (Omura et al., 1997). Based on biochemical evidence, similar inter-
actions occur between other tyrosine kinase receptors, such as SCF and
VEGF receptors and members of the Eph family, and their ligands (Barleon
et al., 1997; Blechman et al., 1995; Lackmann et al., 1998). Furthermore,
structural evidence for this type of interaction has been provided in the case
of FGF receptor-1 and the growth hormone receptor (De Vos et al., 1992;
Plotnikov et al., 1999).
The possibility of blocking PDGF signaling by targeting receptor:receptor
interactions has been demonstrated through the use of Ig-like domain 4 mon-
oclonal antibodies and soluble Ig-like domain 4 (Lokker et al., 1997; Omu-
ra et al., 1997; Shulman et al., 1997). The antibodies blocked PDGF-induced
DNA replication at a concentration as low as 1 nM. The soluble Ig-like do-
main 4 blocked PDGF-induced receptor tyrosine phosphorylation when used
at micromolar concentrations. Although none of these antagonists have yet
been used in vivo, they provide evidence that epitopes mediating receptor:
receptor interactions are candidate targets for future PDGF antagonists.
3. ANTAGONISTS BLOCKING PDGF RECEPTOR KINASE
Low molecular weight tyrosine kinase inhibitors constitute a novel class
of drugs with large potential (reviewed in Druker and Lydon, 2000; Klohs
et al., 1997; Levitzki and Gazit, 1995). It was originally considered that se-
lectivity would be difﬁcult to obtain as most, if not all, of these compounds
act by competing with ATP binding to the relatively well-conserved ATP-

binding cleft of tyrosine kinases. Advances have, however, supported the no-
tion that selective compounds indeed can be generated. Cocrystallization of
tyrosine kinase inhibitors with their tyrosine kinase targets has demonstrat-
ed that these compounds will form contacts with up to 18 amino acids dis-
tributed over the active site of the tyrosine kinase domain, thus allowing for
high speciﬁcity (Mohammadi et al., 1998).
Since 1995, a number of selective PDGF receptor kinase inhibitors have
been characterized, and six compounds—CGP53716 (Buchdunger et
al., 1995), STI-571 (Buchdunger et al., 1996) Ki6896 (Yagi et al., 1998),
RPR101511A (Bilder et al., 1999), AG1296 (Kovalenko et al., 1994), and
SU101 (Shawver et al., 1997)—have demonstrated in vivo efﬁcacy in vari-
ous animal disease models. For all these six drugs, the characterization
of target selectivity remains incomplete. It is, however, clear that none of
these compounds distinguishes between PDGF ␣ and ␤ receptor kinases. The
PDGF receptor kinase inhibitor STI-571 illustrates the somewhat unpre-
dictable cross-reactivity of these types of antagonists; this compound, which
is active against the PDGF receptor at concentrations more than 100-fold
lower than those required to block the tyrosine kinase activity of the recep-
tors for EGF, insulin, or IGF-1, inhibits the structurally less related cyto-
plasmic tyrosine kinase c-Abl with similar potency as the PDGF receptor ki-
nase (Buchdunger et al., 1996; Druker and Lydon, 2000).
4. GENETIC APPROACHES THAT INTERFERE WITH PDGF
RECEPTOR SIGNALING
The fact that both the PDGF ligand and the active signaling receptor oc-
cur as dimers has allowed the development of dominant-negative forms of
PDGF and PDGF receptor that, after different methods of gene transfer, have
been used to block PDGF signaling. Dominant-negative forms of PDGF in-
clude variants that do not undergo processing (Mercola et al., 1990) or that
are altered in the receptor-binding site (Vassbotn et al., 1993), as well as
forms that promote the formation of unstable PDGF dimers that are rapid-
ly degraded (Mercola et al., 1990). In each case, inactive heterodimers be-
tween wild-type and mutated PDGF are formed. In the case of PDGF recep-
tors, a variant with a truncated intracellular domain has been used and
shown to have a dominant-negative effect (Ueno et al., 1991).
Another genetic approach involves the adenoviral-mediated transfer of a
gene encoding the PDGF ␤ receptor extracellular domain with subsequent
PDGF antagonistic effects in a rat model of restenosis (Deguchi et al., 1999).
In a similar model of restenosis, therapeutic effects were also obtained after
local treatment with antisense oligonucleotides targeting PDGF ␤ receptor
expression (Sirois et al., 1997).

B. PDGF Antagonists in Treatment of Cancer
The availability of PDGF antagonists has allowed initial investigations of
the effects of inhibition of PDGF signaling in various tumor cell lines. The
small organic molecule leflunomide (SU101), which acts as a PDGF recep-
tor kinase inhibitor, was shown to inhibit the in vivo growth of a panel of
glioma, ovarian, and prostate cancer cell lines that express PDGF receptors;
however, the growth of other cell lines without PDGF receptor expression
was not affected (Shawver et al., 1997). Also, low passage cultures of tumor
cells derived from glioblastomas developing after an intracranial injection of
PDGF B chain encoding retroviruses showed a reduced growth rate in vitro
when cultured in the presence of PDGF receptor kinase inhibitors (Uhrbom
et al., 1999). Reduction in the growth rate of glioma cells has also been
demonstrated after treatment with dominant-negative forms of PDGF and
PDGF receptors (Kaetzel et al., 1998; Shamah et al., 1993; Strawn et al.,
1994; Vassbotn et al., 1994b).
The notion that DFSP tumors might be treatable with PDGF antagonists
is supported by the observation that the transformed phenotype of collagen
1A1/PDGF B fusion gene expressing cells can be reverted by the use of se-
lective PDGF receptor kinase inhibitors (Shimizu et al., 1999). Interestingly,
primary cultures of cells derived from DFSP tumors show sensitivity to PDGF
receptor kinase inhibitors when grown in vitro or as subcutaneous tumors
in nude mice (Shimizu et al., unpublished observations). Furthermore, a de-
pendency of PDGF ␤ receptor signaling in CMML is indicated by the ther-
apeutic effects of PDGF receptor kinase inhibitors in mice transplanted with
clonal tumor cells derived from Ig-TEL/PDGF ␤ receptor transgenic mice
(Thomasson et al., 1999).
It is well documented that most solid tumors display an increased intersti-
tial fluid pressure (Jain, 1987), and it has been suggested that pharmacolog-
ical reduction of the interstitial fluid pressure might be a way to increase drug
uptake in tumors and thereby obtain better therapeutic effects (Jain, 1998).
A role of PDGF receptor signaling in regulation of the interstitial fluid pres-
sure of tumors was provided by the observation that 4-day systemic treat-
ment with the selective PDGF receptor kinase inhibitor STI-571 reduced tu-
mor interstitial fluid pressure in a rat tumor model of a subcutaneously
grown colon adenocarcinoma (Pietras et al., 2000). The reduction in tumor
interstitial fluid pressure occurred without any effects on tumor size or mean
arterial blood pressure. Interestingly, this treatment also increased transcap-
illary transport into the tumor of a radioactive tracer compound. The effects
on interstitial fluid pressure and tumor uptake were mediated by the target-
ing of PDGF receptors on stromal and perivascular cells, as tumor epithelial
cells in this tumor model are devoid of PDGF receptors. Given the frequent
occurrence of paracrine PDGF stimulation of stromal cells, this potential ap-

plication of PDGF antagonists might be applicable to a large group of solid
tumors.
C. PDGF Antagonists in Treatment of Atherosclerosis
Circumstantial evidence supports a role for PDGF in the development of
atherosclerosis. Moreover, the observation that PDGF BB antibodies signif-
icantly reduced the development of aortic lesions in rabbits fed a cholesterol-
enriched diet provides strong experimental support for the notion that PDGF
stimulation contributes to the formation of cholesterol-induced atheroscle-
rosis (Rutherford et al., 1997a). In addition, the effects of various forms of
PDGF antagonists on the more short-time process of injury- or transplanta-
tion-induced neointima formation have been studied extensively in rat,
porcine, and primate models.
In a pioneering study by Ferns et al. (1991), neutralizing PDGF antibod-
ies were used in the rat carotid artery injury model of neointima formation.
Using an endpoint of 7 days postinjury, a 50% reduction in intimal area was
observed. Similar effects have been described more recently, using the same
animal model, by treatment with 2-bromomethyl-5-chlorobenzene sulfo-
nylphtalimide (Mullins et al., 1994), PDGF ␤ receptor antisense oligonu-
cleotides (Sirois et al., 1997), PDGF receptor tyrosine kinase inhibitor
CGP53716 (Myllärniemi et al., 1997, 1999), PDGF SELEX aptamers (Lep-
pänen et al., 2000a), or adenoviral-mediated transfer of a gene encoding a
soluble form of the PDGF ␤ receptor (Deguchi et al., 1999).
Two studies have also investigated the effects of PDGF antagonists in
porcine models of restenosis. Local intravascular delivery of the tyrosine ki-
nase inhibitor AG1295, impregnated on nanoparticles, was shown to reduce
the intima/media ratio in a femoral artery balloon injury model when ana-
lyzed 4 weeks after injury (Banai et al., 1998). Another PDGF receptor-
selective tyrosine kinase inhibitor, RPR101511A, also showed protective ef-
fects, after systemic administration, in a study where neointima growth was
provoked by deendothelialization of coronary arteries and analyzed 28 days
after injury (Bilder et al., 1999).
Finally, PDGF receptor monoclonal antibodies have been used in baboon
models of restenosis. In one study, restenosis was induced by endarterecto-
my of the carotid artery and balloon dilatation of femoral arteries (Giese et
al., 1999). Six-day treatment with the PDGF ␤ receptor monoclonal anti-
body led to a 37 and 45% reduction in lesions of the carotid and femoral ar-
teries, respectively. No effect was observed after treatment with PDGF ␣ re-
ceptor monoclonal antibodies. The second study evaluated the effects of a
mouse/human chimeric PDGF ␤ receptor antibody together with heparin af-
ter balloon angioplasty of the saphenous artery (Hart et al., 1999). Bolus

treatment with antibody, distributed over 22 days after injury, together with
a continuous infusion of heparin, led to a 40% reduction in the intima/me-
dia ratio.
As mentioned earlier, neointima formation also occurs in association with
transplantation-associated arteriosclerosis. To study the contribution of ac-
tivated PDGF receptors in this process, the effects of the PDGF receptor ty-
rosine kinase inhibitor CGP53716 were studied in a model where cardiac
and aortic allografts from dark agouti rats were transplanted heterotopical-
ly into Wistar–Furth rats (Sihvola et al., 1999). Treatment with the PDGF
antagonist reduced the development of cardiac and aortic arteriosclerosis,
both with regard to the incidence of coronary artery lesions and the intensi-
ty of allograft coronary artery and aortic lesions.
Accumulating evidence thus suggests pharmacological intervention of PDGF
receptor signaling as a novel approach for the treatment of restenosis, as well
as allograft arteriosclerosis. Reports from early clinical trials are likely to ap-
pear within the near future. Most animal studies indicate that PDGF AB and
BB, acting though PDGF ␤ receptors, are the critical mediators that the cel-
lular process blocked by the PDGF antagonists is smooth muscle cell migra-
tion, rather than proliferation (Ferns et al., 1991; Hart et al., 1999; Jawien
et al., 1992; Leppänen et al., 2000a). It is possible that even better effects
can be achieved by combining PDGF antagonists with other treatments. The
potential of such an approach is indicated by the observation that treatment
with antibodies against PDGF and FGF led to significantly better results than
treatment with PDGF antibodies only in the rat carotid injury model
(Rutherford et al., 1997b). One general concern with most published stud-
ies is the lack of evaluation of the persistence of the beneficial effects of PDGF
antagonist treatment. Evidence that this might indeed be a concern was pro-
vided by the finding that, in the rat carotid model, the therapeutic effects of
the PDGF aptamer after a 2-week treatment did not persist 6 weeks after the
end of treatment (Leppänen et al., 2000a).
D. PDGF Antagonists in Treatment of Lung Fibrosis
The role of PDGF stimulation in transplantation-induced obliterative
bronchiolitis was investigated by treatment with the PDGF receptor tyrosine
kinase inhibitor CGP 53716 using a heterotopic rat tracheal allograft mod-
el (Kallio et al., 1999). Reduced myofibroproliferation was observed, and the
degree of obliterative bronchiolitis was reduced by 50%. The therapeutic ef-
fect was observed in the absence of reduced immune activation, suggesting
that the beneficial effect was obtained by the direct reduction of proliferation
of myofibroblasts. Similarly, intraperitoneal delivery of the PDGF receptor
tyrosine kinase inhibitor AG1296 reduced cell proliferation and collagen
synthesis induced by the intratracheal instillation of vanadium pentoxide in

a rat model of pulmonary fibrosis (Rice et al., 1999). Liposome-mediated
transfer of a gene encoding the extracellular ligand-binding domain of the
PDGF ␤ receptor has also been found to ameliorate bleomycin-induced lung
fibrosis (Yoshida et al., 1999).
E. PDGF Antagonists in Treatment
of Glomerulonephritis
PDGF antagonists of three different kinds, PDGF antibodies, PDGF
SELEX aptmers, and receptor tyrosine kinase inhibitors, have all shown pro-
tective effects in the anti-Thy-1-induced rat model of proliferative glomeru-
lonephritis (Floege et al., 1999b; Johnson et al., 1992; Yagi et al., 1998).
Characterization of the lesions after treatment has demonstrated effects on
various aspects of the disease process, including reduced mesangial cell pro-
liferation and matrix accumulation, as well as reduced glomerular macro-
phage influx.
VII. FUTURE PERSPECTIVES
Studies have provided insights into the mechanism of action and in vivo
function of PDGF. Parallel studies have shown that overactivity of PDGF oc-
curs in several different diseases characterized by excessive cell growth, in-
cluding cancer. In several diseases, evidence for a causative role of PDGF in
the pathogenesis has been provided. Components along the PDGF signaling
chain are therefore interesting targets for drug discovery.
Several different types of PDGF antagonists have been described and shown
to act in vivo. Some antagonists act extracellularly by binding to PDGF or
to PDGF receptors, thus preventing ligand–receptor interaction. The ad-
vantage of such antagonists is their specificity; it is even possible to inacti-
vate signaling via one of the PDGF receptors but not the other. The disad-
vantage is that the only available antagonists of this kind are high molecular
weight molecules, i.e., antibodies, ligand-binding domains of PDGF recep-
tors, and SELEX molecules, which are expensive to make and difficult to ad-
minister. A challenge for the future will be to find low molecular weight mol-
ecules that can inhibit the binding of PDGF to its receptors. This may not be
easy, as protein:protein interactions encompassing large surface areas are dif-
ficult to inhibit with low molecular weight molecules. Knowledge about the
three-dimensional structure of the ligand:receptor complex will help in these
efforts.
Other antagonists act intracellularly by inhibiting the PDGF receptor ki-
nase. Their advantage is that they are low molecular weight compounds that

are relatively inexpensive and easy to administer. Their disadvantage is that
they are not specific for PDGF receptor kinases, but inhibit other kinases to
varying extents. This is not surprising given the fact that all efficient kinase
inhibitors target the ATP-binding site that is conserved among kinases. De-
spite this fact, it has been possible to develop fairly selective kinase inhibitors.
An important future goal will be to develop even more specific PDGF re-
ceptor kinase inhibitors, preferentially such that distinguish between ␣ and
␤ receptor kinases. This will require crystallization of the receptor kinases
with and without candidate inhibitors and subsequent chemical optimiza-
tion of the lead compounds.
In the context of cancer treatment, PDGF antagonists have predominant-
ly been envisioned to be of use in the blocking of autocrine growth stimula-
tion. However, an additional interesting possibility is that PDGF antagonists
might be beneficial by inhibiting interactions between tumor cells and stro-
mal fibroblasts and pericytes and endothelial cells, as well as interference
with different aspects of metastasis.
When using PDGF antagonists, it is important not to inhibit important
normal effects of PDGF. The important role of PDGF during embryogenesis
may not be too much of a concern except during pregnancy. Because topi-
cally added PDGF stimulates wound healing, it will be important to investi-
gate whether the inhibition of PDGF slows down wound healing. Another
concern may be the potentially important feedback inhibition on platelet ag-
gregation exerted by PDGF released from platelets. Because this effect is me-
diated via PDGF ␣ receptors on platelets and because overstimulation of the
␤ receptor is implicated in atherosclerosis, fibrosis, and certain forms of can-
cer, it may be advantageous to use antagonists that specifically inhibit sig-
naling via the ␤ receptor and that do not inhibit the ␣ receptor.
The first clinical trials with PDGF antagonists have just started (Eckhardt
et al., 1999) and the results are anxiously awaited. Given the promising re-
sults from experiments using animal models, it is likely that PDGF antago-
nists will be clinically useful in the treatment of certain diseases.
ACKNOWLEDGMENTS
We thank Ingegärd Schiller for valuable help in the preparation of this manuscript.
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—two dim blobs across the gully. For just that second they were
visible as they rose up out of a hollow. A man; and the slighter
figure with him seemed that of a girl. Her hair, glistening like spun
metal in the dim light, hung over her shoulders.
The two figures were struggling. There was the sound of the girl's
low cry, and a grunt from the man.... My low admonition stopped
Jan from firing and in another second the shapes across the gully
had vanished.
"That girl," I murmured. "She tried to keep him from killing us.
Seemed that way, don't you think?"
"Well—"
We waited. From across the gully there was no sound. I could see
now that there was a little ridge in the broken, littered gully floor,
behind which the two figures had vanished. A lateral depression was
there, with the ragged, broken cliff-wall some ten feet behind it.
"Do you suppose there's only one of them?" Jan whispered. "One
man—and that girl—"
"And that—that Thing in flames—"
There was no sign of the animal-like creature. For another moment
we crouched tense, peering, listening. A loose stone the size of my
fist was here beside us. I picked it up. It was weirdly heavy for its
size. Then I flung it out into the gully to the right of us. It fell with a
clatter.
Our enemy was there all right. An arrow whizzed in the darkness
and struck near where the stone had fallen.
Jan laughed with contempt. "Dumb enough—that fellow. Bob, listen,
we've got flash-guns. That fellow with no brains—and just with
arrows—"

True enough. "You stay here," I whispered.
"What's the idea?"
"You wait a couple of minutes. Then throw another stone off to the
right—about the same place. Understand?"
"No, I don't."
"Well, you do it, anyhow."
There seemed a line of shadow to the left of us, a shadow which
extended well out into the gully. The ground dropped down in that
area—a slope strewn with crags, broken with little crevices.
Crouching low, I crept to the bow of the ship, to the left away from
Jan; sank down, waited. There was no sound; evidently I had not
been seen. I started again, picking my way down the slope.
A minute. I was well out into the gully now, ten feet or so down, so
that I could not see the wrecked ship where Jan was crouching.
From here the opposite cliff-wall showed dark and ragged.
Occasionally it yawned with openings, like little cave-mouths. The
place where the figures had been crouching should be visible from
here. The broken, lower side of the little ridge behind which they
had dropped was in view to me now. It was dark with shadow, but
there seemed nothing there.
Slowly, cautiously, I crossed the gully. Two minutes since I had left
Jan? I melted down beside a rock, almost at the edge of the cliff-
wall. And then, out in the gully, far to the right, I heard the stone
clatter as Jan threw it.
There was no answering arrow-shot this time.... One can be very
incautious, usually at just the wrong moment. I recall that I stood up
to see better, though I flattened myself against a boulder. And
suddenly, close behind me, I was aware of a padding, thudding
rhythmic sound on the rocks. I whirled. I had only a second's vision
of a dark bounding animal shape coming at me. My sizzling little
flash went under it as it rose in one of its bounding leaps.

I had no time to fire another shot. Frantically I pulled the trigger-
lever, but the gun's voltage had not yet rebuilt to firing pressure.
Futilely I flung the gun into the creature's face as it bore down upon
me.
The impact of the dark oblong body knocked me backward so that I
fell with it sprawling, snarling upon me. In the chaos of my mind
there was only the dim realization of a heavy body as big as my
own; spindly legs, like the legs of a huge dog. There seemed six or
eight legs, scrambling on me.
Wildly I fought to heave it off. There was a face—a ring of glaring
green eyes; fang-like jaws of a long pointed snout which opened,
snarling with a gibbering, gruesome cry. I shoved my left forearm
into the jaws as they came at my face. They closed upon my arm,
ripping, tearing.
But somehow I was aware that I had lunged to my feet. And the
Thing reared up with me. It was a Thing almost as heavy as myself.
My left arm had come loose from its jaws and as its scrambling
weight pressed me I went down again. A Thing of rubber? It
seemed boneless, the shape of it bending as I seized it. A
gruesomely yielding body. My flailing blows bounded back from it.
Then I knew that I was gripping it by the head, twisting it. The
snarling, snapping jaws suddenly opened wide with a scream—a
scream that faded into a mouthing gibber, and in my grip the Thing
went limp. I cast it away and it sank to the rocks, quivering.
For an instant I stood panting, trembling with nausea sickening me.
On my hands the flesh of the weird antagonist was sticking like
viscous, gluey rubber. Hot and clinging. Hot? I stared at my hands in
the dimness. For a second I thought it was phosphorescence. Then
yellow-green wisps of flame were rising from my hands. Frantically I
plunged them into my jacket pockets. The tiny flames were
extinguished. I stripped off my jacket, flung it away and it lay with a

little smoke rising from it where the weird stuff was trying again to
burst into flame.
The skin of my hands was seared, but the contact with the flames
had been only momentary and the burns were not severe. It had all
happened in a minute or two. I recall that I was standing trembling,
staring at the yawning mouth of a cave entrance which was nearby
in the cliff-face. A movement in there? A moving blob? Then I was
aware that there was a light behind me. Off across the gully there
was a blob of light-fire. A red-green blob, swirling, scrambling. And
the sound of a distant, gibbering snarl....
The singing whizz of an arrow past my head made me turn again.
My human adversary! I saw him now. He was coming at a run from
the mouth of the cave—a wide-shouldered, grotesquely-shaped man
with a brown hairy garment draped upon him. He swayed like a
gorilla on thick bent legs. In one hand he held what seemed an
arrow-sling. In the other he carried a long narrow segment of rock,
swinging it like a club. He was no more than ten feet from me. In
the dimness I could see his huge round head with tangled, matted
blank hair. As I whirled to meet him, his voice was a bellow of
guttural roar, like an animal bellowing to intimidate its enemy.
I turned, jumped sidewise. And abruptly from a rock-shadow another
shape rose up! Slim, small white body, brown-draped with long,
gleaming tawny hair. The girl! Her voice gasped,
"You run! He kill you! In here—this way—"
The bellowing savage had turned heavily in his rush and was
charging us. In her terror and confusion the girl gripped me, shoving
me toward the cave. As we ran I flung an arm around her, lifting her
up. She weighed hardly more than a child. Then we were in the
blackness of a tunnel-passage. I set her down.
"Lie down. Be quiet," I whispered vehemently. She understood me;
she crouched back against the side wall. There seemed a little light
here, a glow which I realized was inherent to the rocks, like a vague,
faint phosphorescence. But it was brighter outside. The charging

savage had evidently paused at the entrance. As I stared now, his
bulky figure loomed there, grotesque silhouette. Then doubtless he
saw me. With another bellow he came charging in.
I stood waiting, like a Toreador, in front of a heavily charging bull. It
was something like that, for as he rushed me, swinging his club and
plunging with lowered head of matted hair, nimbly I jumped aside. I
had seized a rock half as big as my head. He had no time to turn
and poise himself as I jumped on him, crashing the rock at the side
of his broad ugly face as he straightened and swung around.
Ghastly blow. His face smashed in as the rock seemed to go into it.
For a second his hulking body stood balanced upon the crooked legs
and broad flat bare feet. Gruesome dead thing with the face and top
of the head gone, it balanced on legs suddenly turned rigid. Then it
toppled forward and thudded against the passage wall, sliding
sidewise to the ground where it lay motionless.
In the phosphorescent dimness, I dropped beside the girl. She was
panting with terror, shuddering, with her hands before her face.
"It's all right," I murmured. "Or at least, maybe it isn't all right with
you, but he's dead, anyway."
Utterly incongruous, the delicately formed bronze-white girl—and
that hulking, grotesque, clumsy savage.
"Oh—yes," she murmured. "Dear—yes—"
"You speak English—strange, here on Vulcan—"
"But from your Captain Roberts—he was the fren' of mine—of all the
Senzas—"
"He's dead. An arrow in him—lying over there by his wrecked ship—
the rest of them, dead inside—"

"Yes. I know it. That was these Orgs. I was caught—just the last
time of sleep. Tahg—surely it seems it must be Tahg who sent this
Org to take me from my father's home—"
A captive! And she had fought with her savage captor to stop him
from sending an arrow into me. Then, in his absorption as he tried
to stalk me, she had broken loose from him.
"Just this one Org?" I murmured. "Is he the only one around here?
He and that—animal-thing which I killed?"
"That—a female mime—you—you—"
She was huddling beside me, clinging to me, still shuddering. "Two
Orgs there were," she whispered. "And another mime—a fire-male
—"
The flame-creature! Queerly, it was not until that instant that I
thought of Jan. Out there across the gully, that swirling swaying blob
of light-fire! Those snarling sounds! Jan had been attacked by
another of the savages, and by the weird flaming creature! The
mime fire-male, as the girl called it.
I jumped to my feet. "What—what you do?" she demanded.
"You stay here. What's your name?"
"Ama. Daughter of Rohm, the Senza. He my father. He very good
fren' of the Captain Roberts—good fren' of all the Earthmen. Like
you? You are Earthman?"
"Yes. Now Ama, listen—I came here with another Earthman—with
two others, in fact. One of them is over there by the Roberts' ship....
You wait here—"
"No!" she gasped. I had dashed toward the tunnel entrance, but I
found her with me. "No—no, I stay with you."
From the entrance the gully showed dim and silent. Over the little
rise of ground, just the top of the Roberts' spaceship was visible.
Ama clung to me. "I stay with you," she insisted.

Cautiously we picked our way across the gully, up the small
ascending slope. No sound; nothing moving. But now there was a
pungent, acrid chemical smell hanging here in the windless air.
"The fire-mime!" Ama whispered. "You smell the fire? Then he was
angry, ready to fight—"
"He fought," I retorted grimly. "I saw it—"
"Look! Look there—"
Her slim arm as she gestured tinkled with metal baubles hanging on
it.... I saw, up the slope, the blob of something lying on the rocks.
Jan! My heart pounded. But it wasn't Jan. The body of one of the
weird oblong animals was lying there. Lying on its side, with its six
legs stiffly outstretched. Ugly hairless thing, like a giant dog which
had been skinned. I could see now that the grey-green flesh had a
greasy, pulpy look. What strange organic material was this? Certainly
nothing like it existed on Earth. Impervious to heat, as the human
stomach tissue is impervious to the action of its own digestive juices.
Evidence of the thing's flaming oxidation was here. Wisps of smoke
were rising from the ground about the slack body.
Had Jan killed it? The ring of eyes above the long muzzle snout
bulged with a glassy, goggling dead stare. The jaws were open, with
a thick, forked black tongue protruding, and green, sticky-looking
froth still oozing out. The teeth were long and sharp, fangs like
polished black ivory protruding from the jaw. The cause of its death
was obvious. A knife-slash had ripped, almost severed its throat in a
hideous wound where green-black viscous ooze was still slowly
dripping, with smoky vapor rising from it.
For a moment, with little Ama clinging to me, I must have stood
appalled at the weird sight of the dead fire-mime. If Jan had fought
and killed it—then where was he now? And where was that other

Org, companion of the clumsy savage I had killed when it had tried
to attack me?
And where was Torrence?
"Your fren'—he did this?" Ama was murmuring.
"Yes, I guess so." I raised my voice cautiously. "Jan—Oh, Jan, where
are you?"
The dark shadowed rocks mocked me with their muffled, blurred
echo of my call. There seemed nothing here alive, save Ama and
me. The wrecked spaceship lay broken and silent on the rocks, with
the gruesome, strewn bodies of the Earthmen in it. And the body of
Roberts still lay here outside, near the bow.
"Jan—Jan—"
Then Ama abruptly gasped, "The Orgs! See them—up there!"
The cliff which was the gully wall, at this point was some fifty feet
high. I stared up to a patch of yellow light which had appeared there
in the darkness. A band of the murderous Orgs! Carrying flaming
torches, a dozen or more of the gargoyle savages stood above us on
the cliff-brink. One stood in advance of them, pointing down at us.
He was the other one, doubtless, who had originally been down here
with Ama. Around them, half a dozen of the huge greenish mimes
bounded, whining with gibbering cries of eagerness.
And in that instant, an arrow came down. I saw one of the savages
sling it from a flexible, whip-like contrivance. The whizzing metal
shaft sang past our heads and clattered on the rocks.
Ama was clutching me. "You come! Oh hurry—they kill us both."

There was no argument about that. I flung a last look around with
the vague thought that I would see Jan lying here. Then I let Ama
guide me. At a run, we headed back down the declivity and
diagonally across the gully. A rain of arrows came down, clattering
around us, but in a moment most of them were falling short.
"Which way, Ama? Where we go?"
"My people—my village—not too far."
"Which way?"
"Through this cliff. There are passages into the lower valley."

"You know the way?"
"Yes, oh yes."
A dark opening in the opposite cliff presently was before us. The
Orgs were coming down the other cliff now; their bellowing voices
and the whining cries of the mimes were a blended babble.
"A storm is coming," Ama said suddenly.
The distant sky over the lower end of the gully was shot now with
weird lurid colors. In the heavy dark silence here around us, a
sudden sharp puff of wind plucked at us, tossing Ama's long tawny
hair.
"This way—" she added.
My arm went around her as another wind-blast thrust us sidewise,
almost knocking her off her feet. Then clinging together, fighting our
way in a rush of wind which now abruptly was a roar, we plunged
into the depths of the yawning tunnel.
III
I must recount now what happened to Jan, as he told it to me when
after a sequence of weird events, he and I were together again.
When I left him crouching there close against the hull of the
wrecked Roberts' ship, he lost sight of me almost in a moment.
There was just the faint blob of me sliding into a shadow; and then
the lowering ground down which I went hid me. Tensely he
crouched, peering across the gully, listening to the heavy silence.
Two minutes, I had said; and then he must throw the rock. His hand
fumbled around, found a sizable rock-chunk. He understood my
purpose, of course—to divert our adversary across the gully at a
moment when I might be close to jump him from the other
direction.

Jan was excited, apprehensive, just an inexperienced boy. Was the
crouching savage with the girl still there across the gully? There was
no sound, no movement. Was it two minutes now?
He flung the stone at last and raised himself up a little with his gun
leveled. The stone clattered off to the right. But it provoked no
whizzing arrow. No sound of me, jumping upon my adversary....
Nothing.... But what was that? Jan stiffened. Distinctly he heard the
sizzling puff of a flashgun shot. My gun! He knew it must be; it was
to the left, out in the gully. And following it there was a low
gibbering snarl. Faint in the distance, but in the heavy silence plainly
audible.
I had been attacked! Jan found himself on his feet, with no thought
in his mind save to dash to me.... He had taken no more than a few
scrambling leaps on the rocks. He reached the brink of the descent.
Far down and out in the gully it seemed that he could see the blur of
something fighting.
His low incautious movement had betrayed him. From behind him
there was a low whistling. A signal! An eager whining snarl instantly
resounded to it. Jan had no more than time to whirl and face the
sounds when a great bounding grey-green shape was on him!
Jan's shot missed it, and the next second the lunging oblong body
struck him. The impact knocked him backward. His gun clattered
away. Then the huge, hairless dog-like thing sprawled upon him, its
slavering jaws snapping. They found his shoulder as he lunged and
the fang-like teeth sank in....
A miracle that Jan could have kept his wits so that he fumbled for
his knife as he fell. But suddenly he got it out, stabbed and slashed
wildly with it as he rolled and twisted on the ground with the
snarling creature on top of him.... And suddenly he was aware that
the thing had burst into flame!
It could have been only a few seconds during which Jan fought that
weird living fire. It was a wild chaos of horror.... Licking, oozing
flames exuding like an aura from the sticky viscous flesh that

horribly sprawled upon him. Monstrous ghastly adversary, with flesh
that seemed now like burning bubbling rubber, stenching with acrid
gas-fumes....
Just a few seconds, then Jan realized that somehow he had broken
loose from the jaws that gripped his shoulder. He tried to scramble
to his feet. The flames searing his face made him close his eyes. He
was holding his breath, choking. His clothes were on fire....
Then the sprawling, lunging body knocked him down again. He was
still wildly, blindly slashing with his knife. Vaguely he was aware,
over the chaos of snapping snarls, that a human voice nearby with
guttural shouts was urging the animal to dispatch its victim. But
suddenly—as Jan's knife-blade ripped into its throat—the snarls went
into a ghastly, eerie animal scream of agony—a long scream that
died into a gurgle of gluey, choking blood-fluid....
Jan was aware that the creature had fallen from him with its flames
dying. On the rocks he rolled away from it, with his scorched hands
wildly brushing his clothes to extinguish them. Then he was on his
feet, staggering, choking, coughing. But his knife, its blade dripping
with an oozing flame, still wildly waved.
And then he was aware that twenty feet away, a heavy, grotesque
man-like shape was standing with a club and arrow-sling. But with
his flame-creature dead and the sight of the staggering, triumphant
Jan waving his flaming knife-blade—the watching savage suddenly
dropped his club and let out a cry of dismay and fear. And then he
ran.
For a moment Jan, wildly, hysterically laughing, went in pursuit. But
in the rocky darkness the fleeing savage already had vanished....
Then reaction set in upon Jan. His burned face and hands stung as
though still fire was upon him. He was still gasping, choking from
the fumes of his smoldering clothes. His eyes, with lashes singed,

smarted, watering so that all the vague night-scene was a swaying
blur.... He found himself sitting down on the rocks....
And then suddenly he remembered me. Where had I gone? What
had happened?...
Vaguely Jan recalled that I had left him and gone across the gully....
Where was I now?... Then he seemed dimly to recall that he had
heard my shot....
In the dimness suddenly it seemed to Jan that he saw me, far up the
gully to the right, up on the cliff-top. For just a moment he was sure
that it was the shape of me, silhouetted against the sky.... The sight
gave him strength. Still staggering, he ran wildly forward.... A
quarter of a mile; certainly it seemed that far. He had crossed the
gully by now. The figure up above had vanished.... Queer. What was
I doing up there? Chasing the savage?...
Jan climbed the little cliff, which was ragged, and lower here than
elsewhere. It led him to the undulating, upper plateau, crag-strewn,
dim under a leaden sky. But there was enough light so that he could
see the distant figure. It was only two or three hundred yards away,
plodding on, apparently not looking back....
Jan ran after it. And then he was calling:
"Bob! You Bob—"
The figure turned. Started suddenly back, and called:
"Is that you? Jan?"
It was Torrence! He came back at a lumbering run now—Torrence,
bare-headed, gun in hand. But he obviously hadn't had any
encounter. His jacket was buttoned across his shirt; he looked just as
he had when Jan had last seen him, out there at the bow of the
wrecked spaceship when Jan had gone inside to join me.
Torrence stared at the burned Jan. "Why—good Heavens," he
gasped. "You—I saw that thing killing you. I was up here—I started
down, but too late—"

"Where's Bob?"
"Bob? Why—he was killed. Burned—like you. I tried to help him—too
late—the damned things—"
The lameness of it was lost on the still-dazed Jan at that moment. I
had been killed! It struck him with a shock. And as he stood
wavering, trembling, Torrence drew him to a rock.
"Too bad," Torrence murmured sympathetically.
"Where—where were you?" Jan said at last. "We came out of the
ship—couldn't find you."
"I was attacked by one of those cursed Things. Like the one that
nearly got you—like the one that killed Bob. I chased it; shot at it
when I got up here. But I shouldn't have come up—then I saw you
and Bob—too late to get back to you. So I was starting for our ship.
It's off this way, not so very far."
For a little time Jan sat there numbed, and Torrence sat
sympathetically, silently beside him.
"When we get back," Torrence murmured at last, "you can put in
your report with mine. We did our best—but there isn't any use now,
us tackling this thing."
Jan must have been wholly silent, thinking of me, dead, burned,
back there in the darkness of the gully.
"You all right now, lad?"
"Yes," Jan said. "Yes—I'm all right."
"When we get back, we ought to get a bonus," Torrence said. "Don't
worry, Jan—I'll see you get plenty. Your report and mine—to tell
them the hazards of this trip—"
"We should go back?" Jan said.

"Yes, certainly we should. Get back to Earth as fast as we can. No
chance of doing anything else—"
Torrence gazed apprehensively around them in the darkness. That
much at least—the reality of his apprehension as they sat there on
the open plateau—that was authentic enough. And Jan also felt that
at any moment one of the flaming creatures might attack them.
"You strong enough to start now?"
"Yes, sure I am," Jan agreed.
They started, picking their way along. Jan tried to remember how far
we three had come from our own ship until we had discovered the
Roberts' vessel.... For ten or fifteen minutes now he and Torrence
clambered over the rocks.
"You think you know the way?" Jan asked at last.
"Yes—or I thought I did." Torrence's tone was apprehensively
dubious. And that, too must have been authentic. Certainly it would
be a desperate plight to be lost here on Vulcan. "It was Bob who
was sure he knew the way back—"
"I think we are all right," Jan agreed. "That big rock-spire off there—
I remember it."
As they progressed, Jan was aware now that the sky behind them
was brightening. They turned and stared at it.
"Weird—" Torrence muttered.
"Yes—some sort of storm. If it's bad—you suppose we ought to take
shelter? It's pretty open up here."
The sky was certainly weird enough—a swirl of leaden clouds back
there, shot now with lurid green and crimson. And suddenly there
came a puff of wind. Then another. Stronger, it whined between the
nearby naked crags. In a little nearby ravine it caught an area of
loose metallic stones, whirled them before it with a tinkling clatter.

"We came through that ravine, coming out this way," Jan said
suddenly. "I'm sure of it."
Torrence remembered it also. Another blast of wind came; and with
it blowing them, they scurried into the ravine. The lurid storm-sky
painted it with a crimson and green glare, so that the narrow cut in
the rocky plateau was eerie. To Jan it seemed suddenly infernal. He
clutched at the larger, far more bulky Torrence as they hurried along
with the wind blasting them.
Loose metallic stones were blowing around them now with a clatter.
Then suddenly the sky seemed riven by a darting, jagged red shaft
of lightning. And then red rain was pelting them.
"Got to find some place," Torrence panted. He had to shout it above
the roar as the wind tore at his words and hurled them away.
"Over there?" Jan gestured. "Looks like a cave."
The sides of the ravine were rifted in many places with vertical
crevices. They headed toward a wider slit of opening which seemed
to lead well back underground. A place of shelter until this storm
passed....
To Jan, what happened then was weirdly terrifying. He suddenly
realized that as they approached the opening, they were being
pulled at it. Into it! A suction, as though somewhere down
underground this storm had created a partial vacuum—a far lesser
pressure so that the air of the little ravine was rushing into it!
Terrified, both of them now were fighting to keep away. But it was
no use. Like wind-blown puffs of cotton they were sucked into the
yawning opening. A sudden chaos of roaring horror. Jan felt that he
was still clutching at Torrence. Then both of them fell, sliding,
sucked forward as a plunger cylinder is sucked through a pneumatic

tube. The ground here in the passage felt smooth as polished
marble.
For how long they plunged forward Jan had no conception. Roaring,
sucking darkness. Then it seemed that there was a little light. An
effulgence; a pallid, eerie glow, like phosphorescence streaming
from the rocks. The narrow passage was steadily widening; and then
abruptly they were blown out into emptiness.
It was a vast grotto, with smooth metallic floor almost level. The
effulgence here was brighter, so that an undulating, vaulted ceiling
glistened far overhead. For a moment the nearer wall was visible,
smooth, burnished metal rock. Eroded by the winds of centuries, all
the rock here was burnished until it shone mirror-like.
The huge pallid interior roared and echoed with the tumbling wind-
torrents seething in it. A lashing cauldron jumbled with eddying
blasts. Jan and Torrence tried to get to their feet. They could see
now that they were far out from the wall—sliding, buffeted,
desperately clinging together, hurled one way and then another.
Bruised from head to foot, panting, gasping in the swiftly changing
pressures, Jan felt his senses leaving him. A numbed vagueness was
on him, so that there was only the suck and roar of the winds and
the feel of Torrence to whom he was clinging. They were lying prone
now—
"Easing up a little—" He heard Torrence's voice as though from far
away. And then he came to his senses to find that he and Torrence
had hit against a wall of the grotto and were clinging to a projection
of rock.
Easing up a little.... The storm outside lessening.... Jan must have
drifted off again; and after another interval he was conscious that
there was only a tossing, crazy breeze in here. It whined and
moaned, echoing from one wall to another so that the pallid, silvery
half-light seemed filled with a myriad gibbering little voices.
And Jan could see now that he and Torrence had been blown into a
recess of the grotto—a smaller cave. The rock formation here was as

though this were the heart of a monstrous crystal—vertical facets of
strata that glistened pallidly.
"We'll have to try and cross back," Torrence said, and in the confined
space his words weirdly echoed, split and duplicated so that there
seemed many little whispering replicas of his words. "Find that
passage where we came in—"
They were on their feet now—suddenly to Jan there was around
them a vast vista of pallid dimness. A glowing, limitless abyss
stretching off into shadowy nothingness, everywhere he looked.
"Why—why," he murmured, "this place—so large—"
Torrence still had his flash cylinder. He fumbled in his jacket pocket,
brought it out. Amazing thing! As he snapped it on, its tiny white
beam showed mirrored in a hundred places of the paneled,
crystalline walls! The blurred image of Torrence and Jan standing
holding each other with their light-shaft before them, duplicated so
that there were a hundred of them everywhere they looked! And
countless other hundreds smaller and smaller in the myriad
backgrounds!
With a startled curse Torrence took a few steps into what seemed
pallid emptiness, and then suddenly his image was coming at him!
Lost! To Jan came the rush of horror that they might, wander in
here, balked at every turn....
Another startled cry from Torrence stuck away Jan's thoughts.
Neither he nor Torrence had time to make a move. There was
suddenly everywhere the duplicated image of a thick, swaying,
gargoyle savage, standing like a gorilla on thick bent legs, with one
crooked arm holding a flaming torch over his head. A myriad replicas
of him everywhere! Was he close to them, or far away? And in which
direction?

In that stricken second the questions stabbed into Jan's tumultuous
mind. Then he was aware of something whirling in the air over his
head—something crashing on his skull so that all the world seemed
to go up into a splitting, blinding roar of light. He felt his legs
buckling under him. There was only Torrence's fighting outcry and
the sound of a guttural echoing voice as Jan fell and his senses slid
off into a blank and black, empty silence....
IV
I go back now to that moment when Ama and I, pursued by the
roaming band of Orgs, plunged into a tunnel passage that led from
the gully, near the wrecked Roberts' spaceship. It was quite evident
that Ama was aware of the dangers of the wind-storms of her little
world. There was a swift air-current sucking into this passage. But it
was not powerful enough to do more than hurry us along. Once,
where the tunnel branched, there seemed an open grotto up a little
subterranean ascent to the right. It glowed with a brighter pallid
light than was here in the passage. I turned that way with an
interested gaze, but at once she clutched at me.
"No—no. In times of the storm, very bad sometimes in places under
the ground."
There seemed no sign of pursuit behind us. "The Orgs—they run
heavy," Ama said when I mentioned it. In the pale opalescent glow
of the tunnel, I could see her faint triumphant smile as she gazed up
at me sidewise. Strange little face, utterly foreign so that upon
Earth, by Earth standards one would have been utterly baffled to
identify her. But it was an appealing face, and now, with her terror
gone, the sly glance she flung at me was wholly feminine.
"Those fire-mimes," I said. "Couldn't they rush ahead of their
masters, trailing us?" I explained how on Earth dogs would do that,

following their quarry by the scent. She looked puzzled, and then
she brightened.
"I remember. The Captain Roberts told us about that. The mimes are
different. The male and female both—they follow what it is they see,
nothing else."
Then she told me about the weird, dog-like creatures. The male,
exuding a scent—if you could call it that—a vapor which in the air
bursts into spontaneous combustion as it combines with the
atmospheric oxygen.
How long we ran through what proved to be a maze of passages in
the honey-combed ground, I have no idea. Several Earth-miles,
doubtless. Several times we stopped to rest, with the breezes
tossing about us as I listened, tense, to be sure the Orgs were not
coming. Then at last we emerged; and at the rocky exit I stood
staring, amazed.
It was a wholly different looking world here. The pallid underground
sheen was gone; and now again there was the dim twilight of the
interminable Vulcan night. From where we stood the ground sloped
down so that we were looking out over the top of a wide spread of
lush, tangled forest. Weird jungle, rank and wild with spindly trees of
fantastic shapes, heavy with pods and exotic flowers and tangled
with masses of vines. Beyond it, far ahead of us there seemed a line
of little metal mountains at the horizon; and to the left an Earth-mile
or so away, the forest was broken to disclose a winding thread of
little river. It shone phosphorescent green in the half light. The storm
was over now, but still the colors lingered in the cloud sky—a
glorious palette of rainbow hues up there that tinted the forest-top.
Ama gestured toward the thread of river. "The Senzas—my people
and my village—off that way beyond the little water. We go quickly.
But we be careful, until we get beyond the water."
"Swim it?"
"We can. But I think I remember where there is a Senza boat hidden
on this side."

She had already told me more of what happened to her. The Senzas,
primitive obviously, yet with an orderly tribal civilization, were the
dominant race here on little Vulcan. The savage Orgs—a far lower,
more primitive type both mentally and physically—in nomadic
fashion, roamed the metal deserts and little stunted forests which
lay beyond the barren regions. They were, at times of religious
frenzy, cannibalistic, with weird and gruesome festival rites which
Ama only shudderingly sketched.
For the most part, the clumsy Orgs and their weird mime-creatures
were kept from the Senza forests. But occasionally they raided,
stealing the Senza women, and roaming the lush forests for food.
There had been, in the Senza village, one Tahg, a wooer of Ama. An
older man, but somehow well liked by the Senza tribal leader.
Repulsed by Ama, he had threatened her—and then he had vanished
from the village; gone hunting, and the Senzas considered that the
Orgs might have killed him.
"But I think it was Org blood in him," Ama said. "I told the Captain
Roberts that—I remember just before he and his men left us to
finish the repairs of their ship—and then we found later that the
Orgs had killed them all."
Tahg, Ama thought, had become the tribal leader of this group of
the Orgs—indulging with them in their gruesome rites.... Then, just
a few hours ago, two Orgs had crept upon Ama as she slept—with
extraordinary daring for an Org, had successfully seized her and
carried her off. Taking her into the Org country, past the Roberts'
spaceship, where they had come upon me, and Torrence and Jan....
"We be careful now," she was telling me as we stood gazing out
over the forested slope. "After a storm it is when the Orgs mostly
roam—the hunting here is better when the little creatures are out
after the water."
The little creatures! Best of the animal foods here on Vulcan.... The
red-storm quite evidently had emptied torrential rain on the forest.

The fantastic trees were heavy with it. Soddenly it dripped from the
overhead branches. And now as we started down the slope, I saw
the little creatures. Insect or animal, no one could have said. A
myriad sizes and shapes of them, from a finger-length to the size of
a cat. Before our advance they scurried, on the ground, scattering
with weird little outcries. Some flew clumsily into the leaves
overhead; others ran up there on the vines, peering down at us as
we passed. We came suddenly upon a pool of rain-water. Greedily a
hundred little orange-green things, seemingly almost all head and
snout, were crowding at the pool, sucking up the water. With eerie,
maniacal little voices they rolled and bounced away at our approach.
This weird forest! Abruptly I was aware that there were places
where the rope-like vines and leafy branches of the underbrush
shrank away from us as we advanced—slithering and swaying little
vines in sudden movement before us. Sentient vegetation. There are
plants on Earth which shrink and shudder at a touch. Others which
snap and seize an unwary insect enemy. But here it was far more
startling than that. I saw a vine on the ground rise up upon its
myriad little tendrils; the pods, like a row of heads upon it were
quivering, puffing. The extended length of it, like a snake slithered
from my threatening tread.
"It fears every human," Ama said. "A strange thing to you
Earthmen?"
"Well, slightly," I commented. "Suppose it—some of this vegetation
got angry—" Fantastic thought, but the reality of it—a looping,
swaying vine over our heads, as thick as my arm—that was a stark
reality. "Would a thing like that attack us, Ama?"
She shrugged. "There is talk of it. But I think no one is ever truthful
to say it really happened."
We were in the depths of the forest now. In the humid, heavy
darkness it was sometimes arduous going. That thread of river—we
could not see it now, but I judged it still must be half an Earth-mile
away. Once we sat down in a little open glade to rest. In the thick

silence the throbbing voice of the forest, blended of the scurrying life
and the rustling vines, was a faint steady hum. Then suddenly I saw
that Ama was tense, alert, sitting up listening. She looked startled,
abruptly frightened.
"What is it?" I whispered.
"Off there—the vines, they are frightened. You hear?"
It seemed that somewhere near us, the vine-rustling had grown
louder. A scurry, mingled with little popping sounds from the pods.
Someone coming? I recall that the startled thought struck me. Then
from a thicket near at hand a group of little creatures came dashing.
They saw us, wheeled and scurried sidewise. I was on my feet,
peering into the shadowed leafy darkness. I thought I heard a low,
guttural voice. Whether I did or not, the whizz of an arrow past me
was reality enough.
A wandering band of the Orgs were stalking us! At the whizz of the
arrow I made a dash sidewise. My gun was gone; I jerked out my
knife. Ama was up, and another arrow barely missed her—an arrow
that came from a totally different direction so that I knew we must
be already surrounded.
"Ama—lie down! Down—"
A woman under some circumstances can be a terrible handicap. She
didn't drop to the ground; she stood gazing around her in terror, and
then she came running at me, clutching me so that I was futilely
struggling to cast her off. Another arrow sang past our heads, and
then from several directions, the Orgs were bursting into the glade.
I tore loose from Ama, but it was no use. Whatever effective fight I
might have put up, it could have brought a rain of arrows which
might, probably would, have killed the girl.
"Quiet," I murmured. "They've got us. No chance to fight."

I stood trying to shield her as in the dimness the Orgs crowded
around us. Ten or more of them, jabbering at us, seizing me and
presently shoving us off through the forest.
Two or three others seemed to join us in a moment; and abruptly
Ama gasped:
"Tahg! There is Tahg—"
The renegade Senza, quite obviously a leader here, shoved past his
jabbering, triumphant men and confronted us. He was seemingly
startled, and then triumphant at seeing Ama here. Then his gaze
swept to me. He was a big, muscular, but slender fellow. He was
clad in a brief brown drape; but his aspect was wholly different from
the heavy, misshapen, clumsy-looking Orgs. His thick dark hair fell
longish about his ears, framing his hawk-nosed, thin-lipped face. And
his narrow dark eyes squinted at me as he frowned.
"Well," he said, "Earthman? New one?" His English was evidently
less fluent than Ama's, but it was understandable enough.
"Yes," I agreed. "Friendly—like all Earthmen."
He had signaled to the Orgs, and two of them had shuffled forward
and taken Ama from me.
"Jus' good time," Tahg said ironically. "Org gods pleased tonight to
have Earthmen—"
Earthmen! The plural! I had little opportunity to ponder it. Roughly I
was shoved onward through the forest, back to where it thinned into
a stretch of metal desert—and beyond that into a new terrain of
stunted, gnarled trees and rope vines on a rocky ground. To me it
was an exhausting march. Ama, with Tahg beside her, usually was
behind me. Once we stopped and food and water were given me.
When we started again, I saw that, at Tahg's direction, one of the
savages had hoisted Ama to his back, carrying her in a rope-vine
sling. Occasionally other small bands of Orgs joined us, until there
were fifty or more of them, triumphantly returning to their village.

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Advances in Cancer Research 80 1st Edition George F. Vande Woude (Editor)

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