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¡¡ Kenneth
H. Falchuk Research Activities (Co-workers:
M. Montorzi, T.S. Dziedzic, J. Contin, M.
Fasshauer, G. Heffron, G. Wagner, A.
Jardien, A. Tyler). Summary
The
dorsalizing cytoplasmic determinant of Xenopus laevis embryogenesis has been
identified, isolated and partially characterized. It
is a lipid that initiates the entire cascade of inducing signals responsible for formation
of dorsal organs, including brain, spinal cord, eyes, etc.
The storage protein in the oocyte and embryo also has been identified. The lipid affects embryonic as well as
non-embryonic cells, particularly human cancer cells.
The latter differentiate in response to the presence of the lipid. The findings point to novel approaches to treat
human cancer cells by inducing their terminal differentiation. Background
Transplanted
dorso-equatorial gastrula tissue induces an ectopic axis in a recipient embryo. This
discovery by Spemann and Mangold established the importance of inductive signals,
denominated the organizer, to normal
dorsal axis formation and morphogenesis. It
is now recognized that there are a number of chemical substance(s) responsible for the
induction signals. They appear at different
embryonic stages. One set of signals,
considered to be the primary evocator, acts very early in embryogenesis to determine
polarity. These particular signals initiate the entire process and
manifest their activity within 30 min after fertilization through the interaction of a
cytoplasmic and a cortical surface determinant. These
evocator partners are maternally derived and act within the dorsal-vegetal zone before the
first cell division to initiate the downstream cascade of mesoderm-inducing signals
required for dorsal axis formation and morphogenesis.
The primary evocator action can be obviated by treatment of the fertilized
oocyte with ultraviolet (UV)-light or destruction of the future dorsal cortical surface. This leads to failure to form dorsal axis and
organs such as the brain, eyes, spinal cord, etc. The
research focus of the group is to identify the chemical nature of and characterize the
biological function(s) of the cytoplasmic and cortical surface determinants of
embryogenesis. Identification
of the Dorsalizing Cytoplasmic Determinant of Xenopus laevis Embryogenesis
The
cytoplasmic dorsalizing component is known to be stable in organic solvents and to be
destroyed by UV irradiation. Using organic
solvents, therefore, we have extracted and purified a lipid from Xenopus laevis oocytes using HPLC techniques. The amount of the lipid in oocytes at different
stages of maturation (stages I to VI) was determined.
During egg maturation, the lipid progressively accumulates and it is stored
within cytosolic yolk platelets. The
platelets localize the lipid to the vegetal hemisphere following fertilization and,
consequently, distribute it asymmetrically in the embryo.
Normally, nearly 60% of the lipid is utilized during the 5-7 hours required
for embryos to progress from pre-cleavage to blastula.
This information was used to study the dorsalizing capacity of the purified
lipid. The strategy was to radiate the embryo
with UV light to produce axis deficiency, deplete the embryo of the lipid, and then
reconstitute the UV ¡¡
treated
embryo with the lipid to rescue embryos from axis deficiency. When embryos are exposed to UV light, the lipid is
depleted prematurely within minutes. Subsequently,
the embryos develop severe axis deficiency, lacking brain, eyes, spinal cord and other
dorsal organs. These embryos, fated to
develop only ventral structures, however, re-acquire their capability to form dorsal ones
solely by replacement of the depleted lipid molecule immediately following exposure to UV
light. This rescue from dorsal axis
deficiency is presumably due to interaction of the newly supplied lipid with the
pre-localized cortical surface determinant in the dorsal-vegetal zone. Therefore, we propose the yolk platelet lipid is
the cytoplasmic constituent of the primary evocator of the embryo early dorsalizing
inductive cascade. Physical
Chemical Characteristics and Structure of the Dorsalizing Cytoplasmic Determinant. The
cytoplasmic dorsalizing determinant lipid has been submitted to a number of analyses by
UV-, IR- mass- and NMR- spectrometries to determine the chemical structure of the
molecule. The molecule exhibits an absorption
maximum at 375 nm and does not absorb below 250 nm. It
is a lipid with a mass of about 582 Da (its exact molecular mass will be reported later). We are performing the following experiments to
determine its chemical structure: IR spectrum, determination of the number of replacements
with deuteriated methanol, the number of double bonds by palladium charcoal saturation
techniques, the presence of terminal acidic groups by negative ion mass spectrometric
analysis, proton and carbon NMR spectra. We
are collaborating in this aspect of the work with Drs. Andrew Tyler from the Mass
Spectrometry Laboratory of the Chemical Laboratories of Harvard University and Gerhard
Wagner of the NMR laboratories at Harvard Medical School. Oocyte
Storage Protein of the Dorsalizing Cytoplasmic Determinant. DCD
has been found to be stored in the yolk
platelets during oogenesis. The proteins in
these organelles have been fractionated and the molecule that binds the DCD has been
identified. The presence of absorption at 375 nm was used to select the yolk platelet
fraction for further purification and analysis of its DCD content. The initial solubilization of yolk platelets
demonstrated that the lipid was present in the soluble fraction containing lipovitellin
and phosvitin. These two proteins have been
resolved and the lipovitellin fraction was shown to exhibit the absorption maximum at 375
nm that characterizes DCD. The lipovitellin
was purified and its total lipids extracted and separated on HPLC. A lipid was found that has identical elution
properties from a C18 reverse HPLC column and mass as DCD.
Therefore, lipovitellin, a zinc protein known to contain 17% lipid by
weight, is the oocyte storage protein for DCD. The
functional significance is under investigation. Studies
of the Pleiotropic Properties of the Dorsalizing Cytoplasmic Determinant
The
dorsalizing cytoplasmic determinant (DCD) of Xenopus
laevis exhibits pleiotropic properties by exerting profound biological effects on
non-embryonic, adult mammalian cells. It
inhibits the proliferative rate of HT29 colon cancer, SW872 liposarcoma and CH1and S49.1 T
and B mouse lymphoblast cells. The decreased
proliferative rate is due to a slowing of the transition from G1 to S phase. The basis for the phenomenon is under study with
the colon cancer cells. These cells
synthesize, and we have detected, a number of the proteins used to regulate the rate of
transition through the cell cycle. We have
identified CD1 and CDK4, the molecules responsible for phosphorylation of RB protein. We also have assays to detect p21 and p27, the
proteins ¡¡
that
inhibit the activity of CD1. We are testing
the effects of DCD on these proteins. In other cases, when CD1 and CDK4 are inhibited or
p21 or p27 are up regulated, G1 to S transition is slowed, as observed with HT 29 colon
cancer cells incubated with DCD. DCD
switches on, or enhances the activities of, genes associated with differentiation of these
cancer cells as well as of normal 3T3-L1 mouse fibroblasts.
HT29 colon cancer cell production of CEA, alkaline phosphatase and triacyl
glycerol increases by thirty-, fifteen- and two-fold, respectively. Once the lipid is removed from the HT29 incubation
flasks, proliferation resumes but at a slower rate than that of untreated cancer cells. Concurrently, HT29 cell CEA overproduction
returns to control levels. The triacyl glycerol production by SW872 cell increases by
nearly three-fold. In the presence of DCD, 3T3-L1 fibroblasts synthesize six-fold more
triacyl glycerol than control cells and nearly 3-fold greater than those incubated only in
the standard adipocyte differentiation medium. Thus,
the frog dorsalizing cytoplasmic determinant induces differentiation of cancer and normal
cells in vitro.
This capability identifies this natural embryo lipid as a potential
agent for terminal differentiation therapy of cancer cells in vivo. Publications Montorzi,
M., Burgos, M.H., Falchuk, K.H. (2000) Xenopus laevis Embryo Development. Molecular
Reproduction and Development.55:75-82. Teaching
a. Harvard
Medical School Dr.
Falchuk continues to devote a major portion of his professional activities to teaching. He is the Director of the Core I and Core II
Clerkship in Medicine at the Brigham and Women¡¯s Hospital. He is on a number of committees at the Medical
School including the Promotions Committee and Clerkship Directors Committee. b. Departmental/Hospital Dr. Falchuk is the
Chair of the Education Council of the Department of Medicine of the Brigham and Women¡¯s
Hospital. In that capacity, he coordinates
all teaching activities of the students, house staff and fellows working closely with the
individuals in the department responsible for each of these groups. He is on the Vice Chair Advisory Board for the
Department of Medicine. In addition, he is the Chair of the Education Committee of the
Brigham and Women¡¯s Hospital and the Co-Chair of the Education Committee of the Partners
Health Care System. In these committees, he is responsible for the management of all
educational functions of the particular departments of the hospitals, their accreditation,
reviews of programmatic structures, teaching methods, evaluations, salaries, etc. c. National/International Dr. Falchuk
continues to be an active member of the Society of Clerkship Directors in Internal
Medicine as well as a member of the International Advisory Committee of the PanAmerican
Federation of Association of Medical Schools (PAFAMS), an organization whose mission is to
focus on changes in medical education in Latin America. |