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MEDICAL BIOPHYSICS
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Robert F. Bonner, PhD, Head, Section on Medical Biophysics Vladimir Kuznetsov, PhD, DSc, Senior Fellow Tatjana Kiseleva, MD, Guest Researcher Mikhail Ostrovsky, PhD, DSc, Guest Researcherb Ben Ettori, BSEE, Postbaccalaureate Fellow Nicole
Mahdi, HHMI Program Student |
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We develop new biophysical and
optical methodologies for biomedical research and clinical applications.
Recently, we have been focusing on refining technological approaches to
characterizing early stages of disease and to developing strategies to
monitor responses to therapy for and prevent the progression of cancer and
age-related macular degeneration (AMD). By further developing Laser Capture
Microdissection (LCM) and of a new automated, direct transfer
microdissection, we are seeking to optimize the isolation of specific
populations of cells from sections of complex tissues with global analysis of
specific molecular changes. We have applied statistical analysis of gene
expression to identify candidate lists for stage-specific molecular markers
within microdissected cell populations, focusing on technical improvements
needed to identify critical but less abundantly expressed genes, proteins,
and lipids. Laser microdissection
technology development Bonner; in
collaboration with Emmert-Buck, Mage, Obiakor, Pohida As the list of expressed human
genes is completed, a major scientific and medical challenge will be to track
the complex molecular events that drive normal tissue differentiation and
progression of pathologic lesions. Improvements in multiplex molecular
microanalysis have made possible the extraction of DNA, mRNA, proteins, and
lipids from tissue biopsies or cytology specimens so that the levels of
hundreds or even thousands of critical molecules can be analyzed. Given that
cells in complex tissue are biochemically and physically affected by
surrounding cells as well as by remote stimuli from greater distances, the
task of analyzing critical gene expression, protein, and lipid patterns in
development, normal function, and disease progression depends on the
extraction of specific cells from their complex tissue milieu. In
collaboration with the NCI, our laboratory invented and developed LCM and
subsequently sought to improve on the resulting commercial laser
microdissection. Our tissue preparation and microextraction protocols allow
LCM to isolate pure populations of specified cells for subsequent multiplex,
microarray analysis. In
parallel efforts, we have been investigating the biophysics of UV-laser
ablation. We have developed a novel UV-microscope–based methodology for
targeted induction of localized (to about one micron) DNA damage in mammalian
cell cultures, thus permitting the observation of the in vivo dynamics
of the recruitment of GFP-labeled DNA-repair proteins (Celeste et al., Nat
Cell Biol 2003;5:675). In UV-laser microdissection, we have demonstrated
the role played by polymer breakdown and ionization in electrostatic
interactions determining target collection efficiency. In collaboration with
the NIAID, we have used nested PCR and DNA sequencing to demonstrate reliable
molecular analysis in LCM-isolated immunolabeled single cells at the
single-molecule level. However, with the use of ablative UV-laser
microdissection systems, we observed a four-fold reduction in efficiency of
specific single-cell analyses as a consequence of cell fragmentation and UV
damage. As we proceed to specific cell isolation and greater knowledge of
molecular changes in pathology, specific molecular targeting will become
increasingly important to ensure rapid, precise collection of subpopulations
not discernible by morphology alone. Laser
microdissection techniques are now widely used in molecular analysis of
genetics and gene expression changes within target cells in complex tissues. However,
in global proteomic and lipid studies without molecular amplification
methods, the quantity of isolated cells sufficient to perform accurate
characterization of less abundant species is problematic, given that the
microscopic visualization, targeting, and isolation in laser microdissection
has a maximal rate of about 1 to 20 cells per second depending on the
cells’ microscopic distribution within the tissues. Recently, in
collaboration with the Emmert-Buck laboratory (NCI) and the CIT at the NIH, we
invented and are now refining an automatic microtransfer technique based on
molecule-specific labeling of cells that does not require user visualization
or microscopic targeting and is capable of much higher throughput rates. The
technique (patent pending) is built on our solid physical understanding of
thermoplastic microtransfer and uses a much simpler device and transfer films
than commercial laser microdissection instruments. Our current prototype is
capable of isolating all specifically immunolabeled cells within a 1 cm2
region of a standard immunostained tissue section in about five seconds,
corresponding to specific separation from approximately 50,000 cells per
second. The technique allows us to exceed the cell separation rates of
standard technologies such as fluorescence-activated cell sorting while
preserving our ability to harvest directly from standard sections of complex
tissues. This new higher-resolution microtransfer method is particularly well
suited to isolating highly dispersed, specific cell populations (e.g., stem
cells or only those neurons in the supra-optic nucleus that express
vasopressin), with the spatial relationships (morphology) among the specific
cells in the tissue preserved on the transfer film. As the technology becomes
more robust, we will seek to integrate the microtransfer with molecular
profiling of specific cells within tissues, including routine proteomic and
lipidomic analyses, particularly for the large number of less abundant
molecular species (lower copy number per cell). Chuaqui RF, Bonner RF, Best
CJM, Gillespie JW, Flaig MJ, Hewitt SM, Phillips JL, Krizman DB, Tangrea MA,
Ahram M, Linehan WM, Knezevic V, Emmert-Buck MR. Post-analysis follow-up and
validation of microarray experiments. Nat Genet 2002;32:509-514. Mage R, Bonner RF, Obiakor
HAT. Microdissection of single or small numbers of cells for analyses of DNA
and RNA by PCR. In: PCR Technology: Current Innovations, 2d ed. Obiakor H, Sehgal D, Dasso
JF, Bonner RF, Malekafzali A, Mage RG. A comparison of hydraulic and laser
capture microdissection methods for collection of single B cells, PCR, and
sequencing of antibody VDJ. Anal Biochem 2002;306:55-62. Parlato R, Rosica A,
Cuccurullo V, Mansi L, Macchia P, Owens JD, Mushinski JF, De Felice M, Bonner
RF, DiLauro R. A preservation method that allows recovery of intact RNA from
tissues dissected by laser capture microdissection. Anal Biochem
2002;300:139-145. Tangrea
MA, Chuaqui RF, Gillespie JW, Pohida TJ, Bonner RF, Emmert-Buck MR.
Expression microdissection: operator-independent retrieval of cells for
molecular profiling. Diagn Mol Pathol 2004;13:207-212. United
States Patents #6,569,639 issued May 27,
2003: Isolation of cellular material under microscopic visualization. Liotta
LA, Buck MF, Weiss RA, Zhuang Z, Bonner RF. #6,420,132 issued July 16,
2002: Precision laser capture microdissection utilizing short pulse length.
Bonner RF, Goldstein SR, Smith PD, Pohida TJ. #6,251,516
issued June 26, 2001: Isolation of cellular material under microscopic
visualization. Bonner RF, Liotta L, Buck M, Krizman DB, Chuaqui R, Linehan
WM, Trent JM, Goldstein SR, Smith PD, Peterson JI. Gene expression
complexity and statistics of gene expression Kuznetsov, Bonner, in
collaboration with Emmert-Buck, Mulshine If
microdissection and molecular analysis can be made clinically practical, the
expression levels of sets of approximately 20 to 100 critical, stage-specific
disease markers within a selected cell population might provide reliable diagnosis
and intermediate endpoints of response to molecular therapies in individual
patients. Our analysis of large gene expression and protein databases
suggests that a significant fraction of all genes is expressed in any
specific cell type and that the levels of gene products universally exhibit a
highly skewed power-law distribution similar to those characterizing many
other complex systems. We have developed mathematical models for the
evolution of such distributions; our models predict the observed distributions
of genes, protein domains, and gene expression in species of increasing
biological complexity. To
follow macromolecular changes during cancer progression within prostate, breast,
lung, and ovary tissues, we have developed, in collaboration with the NCI,
standard procedures for the isolation of normal and pathological cells from
clinical specimens. Using statistical multivariate analysis, we have
developed mathematical algorithms for the selection of “candidate
genes” from cDNA libraries. The candidate genes’ expression
frequencies are strongly correlated with disease progression. We use our
models of the statistics of expression levels in cell populations to identify
genes differentially expressed in cancer progression. To date, our analysis
points to a critical role for many less abundantly expressed genes at a
critical stage of ovarian cancer progression and suggests that, for most
cancers, diagnostic marker sets should include such low-abundance
transcripts. This notion is guiding our research in the statistics of less
abundant gene products and suitable detection methods. Kuznetsov VA. Family of
skewed distributions associated with the gene expression and proteome
evolution. Signal Process 2003;83:889-910. Kuznetsov VA, Knott GD,
Bonner RF. General statistics of stochastic process of gene expression in
eukaryotic cells. Genetics
2002;161:1321-1332. Kuznetsov VA, Pickalov VV,
Senko OV, Knott GD. Analysis of the evolving proteomes: predictions of the
number of protein domains in nature and the number of genes in eukaryotic
organisms. J Biol Syst 2002;10:381-407. Gene expression during
normal development and pathology progression Bonner; in
collaboration with DiLauro, Malekafzali, Mushinski, Parlato We
have applied single-cell LCM capture technology to study gene expression
during normal development (spermatogenesis and thyroid bud development) and
in stage-specific pathological cells (e.g., cancer precursors). In
collaboration with Roberto DiLauro’s laboratory, we examined gene
expression patterns associated with the primordial thyroid and the adjacent
cells from which it derives in both wild-type and knockout mice. In
collaboration with Fred Mushinski, we are applying the same LCM methodology
to study early changes in the well-established plasmacytoma model in BALB/c
mice. Jointly, we have developed robust LCM techniques for isolating specific
cells from mouse cryosections and have recovered good yields of high-quality
mRNA. In collaboration with Life Technologies, Inc., we have made cDNA
libraries of high diversity and message length from 100 ng of microdissected
mRNA (about 5,000 cells). Using Affymetrix microarray hybridization, we can
determine global gene expression profiles in these libraries while preserving
the libraries for isolation of the full-length message of identified
stage-specific genes. We are planning to apply these methods, which have
undergone validation in reproducible animal models, to clinical specimens of
early stages of disease progression for microdissection of critical and rare
cell populations. We foresee an evolution of molecular diagnosis from one
based on the qualitative or quantitative analysis of a few key macromolecules
to one in which special clustering algorithms analyze complex multivariate
databases. Such analyses should permit a more complete identification of
highly correlated clinical cases and allow us to characterize their response
to molecular therapies specifically designed to prevent progression. To this
end, we have refined the LCM of rare cell populations, particularly those
that might be accessible by serial, minimally invasive cell harvesting from
patients. Jones
MB, Michener CM, Blanchette JO, Kuznetsov VA, Raffeld M, Serrero G, Emmert-Buck
MR, Petricoin EF, Krizman DB, Liotta LA, Kohn EC. The granulin-epithelin
precursor/PC-cell-derived growth factor is a growth factor for epithelial
ovarian cancer. Clin Cancer Res 2003;9:44-51. Mutsuga
N, Shahar T, Verbalis JG, Brownstein MJ, Xiang CC, Bonner RF, Gainer H.
Selective gene expression in magnocellular neurons in rat supraoptic nucleus.
J Neurosci 2004;24:7174-7185. Prevention of
progression of age-related macular degeneration through photoprotection Meyers, Ostrovsky,
Kiseleva, Ettori, Mahdi, Bonner; in collaboration with Avetisov, de
Monasterio The
associations of late stages of age-related macular degeneration (AMD) with
cataracts, earlier cataract surgery, cumulative exposure to sunlight, and
pigmentation all support the hypothesis that chronic photochemical injury
drives AMD progression. Lipofuscin accumulates with age in the retinal
pigment epithelium (RPE) and co-localizes with acute photosensitization of
reactive oxygen intermediates (ROIs) in the primate retina. We model the
normal accumulation of potentially damaging photoproducts with age in the RPE
and Bruch’s Membrane (BM) complex and as well as changes induced by
additional spectral filtering of light reaching the macula. Lipofuscin
granules in the RPE contain at least 10 different fluorescent photochemical
products, including A2E (N-retinylidene-N-retinylethanolamine), its epoxides,
and other as yet chemically unidentified A2E-related fluorophores. The
precursors of these fluorophores originate from reactions of all-trans-retinal
within the rod outer segment (ROS) discs during periods associated with
significant rhodopsin bleaching (i.e., normal daylight). Although RPE
lysosomal processing enzymatically digests over 99 percent of the shed ROS
discs, A2E and related fluorophores are not digested but are concentrated
into lipofuscin granules. By age 60, the average concentration of A2E within
RPE cells reaches about 400 microM in normal eyes. However, A2E is toxic to
cellular membranes at much lower concentrations. We hypothesize that
segregation of A2E into lipofuscin granules and prevention of its
redistribution into critical membranes is required for RPE health. We
developed a biophysical model by using normal values of pupil size, lens
transmission, and the rod dark adaptation time constant trh to
determine average retinal spectral irradiance, steady-state concentration of
all-trans-retinal, all-trans-retinal photosensitization of
oxidative damage, all-trans-retinal reactions to form A2E-related
species in ROS, and A2E photo-oxidation within RPE lipofuscin granules as a
function of age and ambient light intensity. Our model predicts a decline of
about one third in the action spectra–weighted short-wavelength macular
irradiance with each decade and a nearly constant production rate of
A2E-related fluorophores in the RPE during the first 60 years (falling
significantly thereafter). A similar age dependence of total lipofuscin
granule volume and total fluorescence per RPE cell was reported recently in
human cadaver eyes. Since the rates of lipofuscin increase with age are
slower than the rate of decrease in short-wavelength macular irradiance in
the phakic eye with age, ROI photosensitization in the RPE should also fall
with increasing age. Photo-oxidative stress in the outer retina might arise
from the smaller amounts of A2E-related fluorophores observed in critical
membranes of the RPE/BM complex. However, if the RPE/BM complex were the site
of photo-oxidative injury driving AMD progression, the magnitude and rate of
the oxidative injury would be expected to increase dramatically with action
spectra–weighted macular irradiance following cataract removal and
intraocular lens (IOL) implantation. Predicting that mechanisms of photo-oxidative
injury decrease with advancing age, we propose a contrarian view that singlet
oxygen generation by RPE lipofuscin allows the chemical alteration of
accumulating A2E, thereby limiting the steady-state levels of A2E ([A2E]ss)
in the RPE, the redistribution of A2E into retinal membranes, and A2E
chemical toxicity. Singlet oxygen generated photochemically within the
lipofuscin granule reacts with its A2E to form A2E epoxides, which then react
to form increasingly complex cross-linked molecules. As short-wavelength
macular irradiance falls with age, the rate of A2E photo-oxidation falls 15-
to 20-fold, causing [A2E]ss to increase in the normal phakic eye
even as rod bleaching and A2E production decrease. Our theoretical model of
macular aging reproduces the normal age dependence of lipofuscin and A2E and
provides a primary cytotoxic mechanism in which, once A2E exceeds a threshold
concentration in the RPE cell, A2E redistribution into membranes causes
damage with or without additional photo-activation. In our model, it is
primarily the yellowing of the lens with age that distorts the original
spectral balance between rate of production and rate of photo-oxidation found
in youth and allows the [A2E]ss to rise. We are evaluating noninvasive
retinal imaging methods that might permit clinical validation of our
predictions of photochemical changes following cataract surgery as well as of
our predictions of the benefits of specific spectral photo-protective
filters. Our proposed spectrally selective “sunglasses” reduce
both rod activation in bright ambient light and the accumulation of toxic
photoproducts in the RPE. In collaboration with Sergei Avetisov, we are
designing clinical studies of the effects of such filters on progression of
both early and moderate AMD following cataract surgery and IOL implantation. Meyers
SM, Ostrovsky MA, Bonner RF. A model of spectral filtering to reduce
photochemical damage in age-related macular degeneration. Trans Am
Ophthalmol Soc 2004, in press. aRetina Consultants, Des Plaines, IL bRussian
Academy of Sciences, Moscow, COLLABORATORS Sergei Avetisov,
MD, Eye Institute of the Russian Academy of Medical Sciences, Francisco de
Monasterio, MD, Office of the Clinical Director, NEI, Roberto DiLauro,
PhD, University of Naples Medical School and Stazione Zoologica Anton
Dohrn, Michael R.
Emmert-Buck, MD, PhD, Laboratory of Pathology, NCI, Rose G. Mage, PhD,
Laboratory of Immunology, NIAID, Arash Malekafzali,
MS, Arcturus Bioscience Inc., James L. Mulshine,
MD, Medical Oncology Branch, NCI, Fred Mushinski,
MD, Laboratory of Genetics, NCI, Harold Obiakor,
PhD, Laboratory of Immunology, NIAID, Rosanna Parlato, PhD,
European Molecular Biology Laboratories, Tom Pohida, MSEE, Computational Biology and
Electronics Laboratory, CIT,
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