News and Publications
The Skaggs Institute For Chemical Biology
Scientific Report 1997-1998
Studies in Molecular and Experimental Medicine
M. Suzuki, J. Johnson, B. Nowakowski, M. Nishizawa, V. Pasquetto, R. Piotrowicz,
A. Demina, E. Beutler
Makoto Suzuki is using the phage-display technique to discover receptors
on prostate cancer cells. This technique is based on the production of tens of
millions of different peptide sequences and selection of the sequences by cultured
prostate cancer cells (Fig. 1).
After 4 cycles of "panning" (absorption and desorption of phage particles
on the target cells), some consensus sequences were recovered from a 12mer peptide
library. No obvious linear sequence similarity was apparent between any of the
selected peptides, a finding compatible with the recognition of different cell-surface
molecules by different peptides. Of 18 clones sequenced, 5 were identical. Interestingly,
1 clone was detected with different methods of eluting phage from cells.
Detection of the peptides that bind to prostate cancer cells provides an
opportunity to identify the receptors to which the peptides bind and to determine
whether these receptors are specific to the prostate, specific to cancer, or
both. If receptors of sufficient specificity are found, they might be used as
targets for tumoricidal drugs; the receptors would attach drug molecules to the
targeting sequences. This targeting of drugs could eventually lead to more effective
treatments of prostate cancer.
In an article in the Journal of Biological Chemistry, Jennifer Johnson
reports the importance of phosphorylation of the p47phox subunit
of NADPH oxidase on serine 303 or 304. The results of her studies suggest that
the effect of phosphorylation is related to either the increase in hydrophilicity
around serines 303 and 304 or that activation involves the formation of a metal
bridge between the phosphorylated serines and another region of the protein.
Dr. Johnson also showed that p47phox-deficient B lymphoblasts
that express the p47phox S(359,370) double mutation have dramatically
lower levels of enzyme activity and phosphorylation of p47phox than
do p47phox-deficient B lymphoblasts that express wild-type
p47phox. In addition, the mutant p47phox does
not translocate to the plasma membrane when the cells are stimulated.
In contrast, normal phosphorylation and translocation occur in mutants containing
aspartate or glutamate at positions 359 and 370, but oxidase activity is still
greatly reduced. These results imply that a negative charge at position 359,
position 370, or both is sufficient to allow the phosphorylation and translocation
of p47phox, but that features unique to a phosphorylated hydroxyamino
acid are required to support production of superoxide.
Barbara Nowakowski is analyzing a fusion protein, PAX3-FKHR, associated with
alveolar rhabdomyosarcoma, a soft tissue tumor in children. This protein is generated
by a chromosomal rearrangement that results in the fusion of the amino-terminal
DNA-binding domains of PAX3 and the carboxy-terminal transcriptional regulatory
domain of the winged helix protein FKHR. PAX3-FKHR, expressed by the avian retroviral
vector RCAS, induces foci of transformed cells and anchorage-independent growth
in cultures of chicken embryo fibroblasts. The fusion protein is a stronger transcriptional
activator than is PAX3. The gain-of-function mutation suggests a potential mechanism
Dr. Nowakowski has begun an analysis of a correlation between transactivation
and transformation in chicken embryo fibroblasts. She has constructed a series
of deletion mutants of PAX3-FKHR to determine the molecular domains required
for transformation of the fibroblasts. Comparisons of DNA binding, transactivation,
and transformation by the various mutant proteins will be important to an understanding
of the method by which the fusion gene alters normal gene expression and causes
Makoto Nishizawa is continuing his studies of oncogenic transcription factors
that belong to the BZip family of proteins. He has done a genetic analysis of
Maf, a protein that plays an important role in the differentiation of hematopoietic
cells. Maf has cancer-inducing potential, and Dr. Nishizawa's genetic work has
uncovered mutations that activate this oncogenicity. He is currently concentrating
on the role of protein dimerization and DNA binding in oncogenic transformation.
For this analysis, he has constructed chimeric proteins in which the dimerization
and DNA-binding domains of Maf have been replaced with corresponding domains
of other, related BZip proteins such as Jun, Fos, and yeast GCN4 transcription
factors. The chimeric proteins differ in their DNA targets and in their ability
to form heterodimers or homodimers. Among these factors, Maf, Jun, and Fos are
all oncogenic, but they induce different tumors: musculoaponeurotic fibrosarcoma,
fibrosarcoma, and osteosarcoma, respectively. The yeast transcription factor
GCN4 is identical with Jun in DNA-binding specificity but is not transforming.
Dr. Nishizawa is now testing the transforming ability of the chimeric molecules
in vitro and the specificity of tumor formation in vivo. The information obtained
with these constructs will be important in identifying downstream target genes
that are upregulated or downregulated by transcription factors of malignant transformation
and may lead to better understanding and treatment of cancer.
Valerie Pasquetto is studying hepatitis B virus (HBV). After antigen recognition,
HBV-specific cytotoxic T lymphocytes (CTLs) induce a necroinflammatory liver
disease in HBV transgenic mice. An early event in this process is the activation
of apoptosis by the CTLs in a fraction of the hepatocytes that are positive for
the HBV surface antigen. In addition, the CTLs inhibit HBV replication by noncytopathically
eliminating viral nucleocapsids from the cytoplasm of the remaining viable hepatocytes
by a process that depends on IFN-Γ and TNF-α.
Dr. Pasquetto's studies were undertaken to determine the fate of cytoplasmic
nucleocapsids and their cargo of replicative intermediates in the apoptotic hepatocytes,
because the elimination of HBV during viral hepatitis traditionally was thought
to require the destruction of infected cells.
Primary cultures of HBV transgenic mouse hepatocytes were incubated in vitro
in the presence or absence of cells from a CD8+ CTL clone specific for cells
bearing the HBV surface antigen. The cultures were harvested at various intervals
to monitor cell survival and capsid-associated HBV replication. Concomitant with
the induction of apoptosis in less than 1 hour and the destruction of all hepatocytes
in less than 4 hours, the capsid-associated replicative intermediates were shifted
from the viable adherent hepatocytes to the apoptotic debris and then to the
culture supernatant in an orderly, time-dependent manner. The capsid-associated
replicative intermediates were stable in the apoptotic cellular debris and culture
supernatant for at least 5 days after the addition of the CTLs, at levels comparable
to those detected in the starting hepatocytes.
These results (1) indicate that HBV capsids and their associated replicative
intermediates survive the apoptotic destruction of the cells that produce them
and (2) confirm that downregulation of HBV replication by CTLs and other cytokine-inducing
stimuli requires viable hepatocytes to be effective. The replication competence,
potential infectivity, and ultimate fate of these capsids in the presence of
macrophages and nucleocapsid-specific antibodies in vivo are under investigation.
Randolph S. Piotrowicz is focusing on the roles of the 27-kD heat-shock protein
(HSP27) in vascular endothelial cell behaviors relevant to angiogenesis in breast
tumors. In particular, he has investigated the effects of HSP27 expression and
phosphorylation on the regulation of cell motility. He showed that enhanced expression
of HSP27 in endothelial cells resulted in greater motility. Expression of a nonphosphorylation
mutant HSP27 retarded cell migration. HSP27 was localized to dynamic F-actin
structures of migrating cells, suggesting that HSP27 regulates the formation
of lamellipodial F-actin and thus regulates cell motility.
In addition, the release of the angiogenic hormone fibroblast growth factor
2 (FGF-2) was studied. A novel mechanism for the release of FGF-2 and the capacity
for high molecular weight forms of this hormone to inhibit cell migration have
been determined. The inhibition is unique because it requires the presence of
the estrogen receptor for transduction of the inhibitory signal induced by high
molecular weight forms of FGF-2. This work suggests that coupling of peptide
and steroid hormone receptors in the transduction of signals by an angiogenic
factor can occur in endothelial cells. This possibility is being investigated.
These studies may be relevant to the spread of breast cancer in women.
Anna Demina studied a mutation in UGT1A1, a gene important in the
disposal of bilirubin, the yellow bile pigment that is the cause of jaundice.
The promoter of this gene may have 6 or 7 thymine-adenine (ta) repeats. The 7-repeat
promoter is less efficient, and persons who inherit this mutation tend to have
a mild form of jaundice known as Gilbert syndrome. Dr. Demina studied the population
genetics of this mutation to determine whether the lower frequency of jaundice
in Africans was due to a lack of the 7-repeat mutation. However, she found that
persons of African ancestry include individuals with 5 or 8 repeats as well as
individuals with 6 or 7 repeats. Even though bilirubin levels are lower in Africans
than in Europeans, the promoters with lower activity were more common. An incidental
finding was a polymorphism at nucleotide 106, tightly linked to the (ta)5 haplotype.
Among persons of Asian ancestry, in whom jaundice is more common, the deficient
(ta)7 form was, surprisingly, less common. Thus, within the
Caucasian ethnic group a strong correlation exists between the number of repeats
in the promoter and bilirubin level; that is, promoters with higher numbers of
repeats are associated with higher levels of bilirubin. However, between ethnic
groups, lower numbers of repeats are associated with higher levels of bilirubin.
Serum levels of bilirubin are influenced by many factors, both genetic and
environmental. These data suggest that the unstable UGT1A1 polymorphism
may "fine tune" the plasma level of bilirubin within population groups, maintaining
a level that is high enough to provide protection against oxidative damage but
sufficiently low enough to prevent kernicterus in infants. The differences in
bilirubin levels that still exist between racial groups, despite changes in the
number of promoter repeats, imply that the changes have occurred sufficiently
recently that full compensation has not yet been achieved.