Gene amplification in mammalian cells: a comprehensive guide

576 Pages·32.728 MB·English

by  Kellems, Rodney E

Gene
Amplification
in Mammalian
Cells

Gene
Amplification
in mammalian
Cells
A Comprehensive Guide
edited by
Rodney E. Kellems
Baylor College of Medicine
Houston, Texas
CRC Press
Taylor & Francis Group
Boca Raton London New York
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Library of Congress Cataloging-in-Publication Data
Gene amplification in mammalian cells: a comprehensive guide / edited
by Rodney E. Kellems.
p. cm.
Includes bibliographical references and index.
ISBN 0-8247-8756-0
1. Gene amplification. 2. Gene amplification—Research-
-Methodology. I. Kellems, Rodney E.
QH450.3.G46 1992
599'.087'322-dc20 92-26048
CIP
Foreword
The experimental study of variable gene copy number has a venerable history,
starting with Sturtevant's studies on the bar locus in Drosophila some 65 years
ago [1]. Gene duplications are common in bacteria under appropriate selection
conditions (see Ref. 2). The demonstration in 1968 of developmentally regulated
amplification of genes, most dramatically shown by Brown and Dawid with
Xenopus ribosomal genes [3], raised the question of whether high-level differen-
tiation specific gene expression was the result of selective gene amplification in
higher eukaryotes. With the advent of molecular biology in the mid-1970s, this
question was answered with an essentially uniform "no."
Gene amplification remained a "dormant" subject as applied to mammalian
cells until 1978-1979 when documentation of gene amplification as a mechanism
for acquired resistance in cultured mammalian cells employing molecular biology
techniques by the Schimke [4] and the Stark [5] groups opened a new era for the
study of gene amplification. It should be noted, however, that their definitive
studies were presaged by the cytogenetic studies of Beidler and Spengler with
HSRs (homogeneously staining chromosome regions) in methotrexate-resistant
Chinese hamster lung cells [6] and of Levan et al. with minute chromosomes of
Sewa cells [7]. The finding of gene amplification was initially surprising, inas-
much as both research groups had originally assumed that enzyme overproduction
was going to be the result of an "up/promoter" mutation. More recently, a large
number of cases of gene amplification in cultured mammalian cells have been
found, examples of which are described in this volume. One gets the impression
Hi
iv Foreword
that with appropriate selection protocols, virtually any gene can be amplified,
unless it or a closely linked gene is otherwise detrimental to cell growth or
survival. For instance, we have reported that the trileucyl peptide calpain inhibitor
ALLN, which is hydrophobic, is cytotoxic and resistance can be readily selected
in CHO cells that results from amplification of the MDR1 gene [8]. Recently, we
repeated the selection procedure with the addition of verapamil to prevent function
of the P-170 glycoprotein exit pump and found that resistance develops, which is
associated with overproduction (and gene amplification) of an aldo-ketoreductase,
an enzyme that inactivates the active form of ALLN (R. Sharma et al., in
preparation). Surely the list of amplified genes will continue to grow.
The status of experimental gene amplification in mammalian cells as of 10 years
ago was represented in Gene Amplification, the outcome of a conference held at the
Banbury Conference Center of the Cold Spring Harbor Laboratories in 1981 [9].
This current volume, edited by Rodney Kellems, is a welcome updating of the
status of the field. Many of the current contributors were authors in 1982, many are
scientific "offspring" of those earlier contributors, and still others are new,
vigorous contributors.
What follows are a few brief thoughts about where gene amplification has gone
from those early days and where it might be going.
I. AMPLIFICATION IN INDUSTRY
It is surprising to me how rapidly the phenomenon of gene amplification was
pursued within a commercial context and what a major role it has played as a
means to produce certain proteins for medical usages where bacteria or yeast do
not make biologically active proteins and where mammalian cells do. The fact that
DHFR amplification (methotrexate resistance) in Chinese hamster ovary cells is
the most commonly employed technique commercially is in some part a historical
accident of the time (the early 1980s) because scientists who joined various
companies were familiar with this system. Clearly, this is an example of extremely
rapid technology transfer from the laboratory to industry. It is also important to
note that scientists associated with the industry are making important and basic
contributions to the field.
II. AMPLIFICATION IN CANCER BIOLOGY
Prehaps the major area in which gene amplification is observed in the "real world"
is in cancer biology. Interestingly, although there are examples of gene amplifica-
tion in clinical resistance (DHFR and methotrexate), it does not appear that gene
amplification is a common mechanism for clinical cancer resistance, as opposed to
its common occurrence as studied in the laboratory. Why this difference exists is
clearly worthy of understanding in the future. On the other hand, oncogene
Forewordh v
amplification is extremely common. From the time of initial discovery of gene
amplification in mammalian cells, various predictions of a role for amplification in
carcinogenesis were made [10-13]. Starting in the mid-1980s, reports of oncogene
amplification commenced, and now amplification of a variety of oncogenes has
become a common, but certainly not universal, theme, with respect to either the
cancer type, the oncogene involved, or the percentage of a specific cancer type
with a specific oncogene amplification.
Perhaps there are two extreme views of oncogene amplification and cancer. One
assertion would be that amplification of an oncogene is a necessary and/or a
sufficient event(s) to result in a cancer cell. Ancillary to this view might be a
relationship between the copy number of an amplified gene and tumor behavior. In
the instances of experimental gene amplification and drug resistance, the correla-
tion between gene copy number and the biological response of cells, i.e., the
degree of resistance, is fairly good. However, in the case of oncogene amplifica-
tion, such simplistic relationships do not appear to hold. The other extreme view
would be that oncogene amplification has no role in the malignant process and is a
neutral manifestation of an underlying state of a malignant cell that generates a
high degree of genetic instability, of which gene amplification is one, but by no
means the only, type of genomic alteration found.
Probably there is no simple and single answer, and the understanding will lie
somewhere in the middle of these extremes, depending on the oncogene and the
stem-cell type involved, including the unique logic of control of proliferation and
differentiation of that cell type. Most thinking about gene amplification assumes
that overproduction of the oncogene product is the casual end-point. However, an
alternative consideration for oncogene amplification might involve binding to
amplified DNA segments of regulatory proteins present in limited amounts in
cells, thus reducing their availability for regulation of other genes and thereby
altering cell regulatory behavior unrelated to expression of an oncogene per se.
This type of mechanism may be involved in instances where the degree of
oncogene amplification vastly exceeds any copy number that may seem reason-
able, based on drug resistance amplifications at clinically reasonable drug levels
(say 5- to 10-fold at most).
III. GENE COPY DIFFERENCES AMONG INDIVIDUAL
HUMANS
Another area where variable gene copy may play a more significant role than
currently understood is in individual responses to various environmental agents.
It is most often assumed that all individuals have one copy of a gene per haploid
genome. Unless an investigator is highly sensitive to looking for copy number
differences, such differences will be overlooked in standard use of restriction
fragment analyses. However, there are some examples where gene copy number
vi Foreword
differences have been reported. For instance, Ohlsson et al. [14] reported a family
with an additional copy of a beta-interferon gene that was inherited as a Mendelian
trait. What might constitute a selection for this amplification is not clear. Prody
et al. [15; also Soreq et al., this volume] reported a father and son with complex
amplification of a "true" cholinesterase gene and suggested that chronic exposure
to the insecticide parathion may have played a role in selection of this amplification
event(s). More recently, Ingelman-Sundberg and his colleagues [16] have reported
in preliminary form a family with 10- to 15-fold amplification of the CYP2D gene
(a member of the P-450 drug-metabolizing gene family), detected by virtue of lack
of response to certain pharmacological agents. Interestingly, the P-450 gene
family constitutes an evolutionary amplification process (a member of a multigene
family), at least one member of which appears to undergo amplification in germ
cell lineages in historical time as well. Thus, the question arises as to how often
differences among individuals, perhaps most readily observed in relation to
responses to drugs, may result from genetically inherited differences in gene copy
number. Perhaps even more interesting are the questions of what exposures, what
selection pressures, and what mechanism(s) have led to the fixation of multiple
gene copies in germ cells in individuals of a species. The realization that variations
in gene copy number occur may lead to new understanding of individual differ-
ences among members of a species.
IV. AMPLIFICATION MECHANISMS
A major effort in the field of gene amplification in mammalian cells in recent years
has involved attempts to understand the mechanism(s) whereby genes are ampli-
fied. Early studies on the definition of amplified DNA segments concluded that a
variable-length chromosome was amplified [17,18]. These studies employed cell
lines subjected to multiple step selections over long time periods and concluded
that multiple recombination events had occurred, within the gene as well as in
flanking regions, and indicated a high degree of genomic "flux." More recent
studies, described in this volume, have focused on the study of cell populations as
soon as possible after the initial amplification event(s) have occurred and have
employed studies of the structure of amplified sequences as well as their localiza-
tion by use of newly available and highly sensitive in situ hybridization tech-
niques. Certain of these types of studies favor some form of sister chromatid
exchange phenomenon as a primary event (see Stark, this volume). An interesting
and rather common structure of amplified arrays of genes is their existence as
inverted (head to head) structures (see Fried et al., this volume). Such structures
are most readily accounted for by aberrant replication and recombination models,
as discussed in this volume (see Fried et al. and Wahl et al. for extensive
discussions). I suspect that the details of how amplified genes are generated and
exactly what segment of a chromosome is amplified will be variable from one cell
Foreword vii
population to another, and that a single mechanism will not account for all
instances of amplification, whether they are a part of the initial, i.e., primary,
event, or as cells are either maintained under constant selection or subjected to
additional selection pressure.
V. AMPLIFICATION OF GENES AS A RESPONSE TO
PERTURBATION OF CELL CYCLE PROGRESSION
I have been struck over the years with the frequency with which cell variants with
amplified genes bear a number of cytogenetic abnormalities in addition to the
cytogenetic consequences of specific gene amplification. These include differing
degrees of aneuploidy (polyploidy) and presence of deletions and translocations,
including extreme cases of the existence of so-called "marker chromosomes,"
which basically constitute breakage and recombination of chromosome fragments
to constitute unrecognizable new chromosomes (see Fougere-Deschartrette et al.
[19] for some examples). Such chromosomal changes, including aneuploidy,
deletions, translocations, and amplifications, are, of course, common cytogenetic
abnormalities of cancer cells. Thus, an understanding of mechanisms of gene
amplification may provide insight into types of genomic alterations (including
rearrangement events) that are so very common in cancer and are not explicable
within the context of the understanding of point mutational events.
Such "global" cytogenetic changes in genome structure suggest that cell
variants selected for a specific amplified gene may have been subjected to events
that variously alter overall chromosome integrity. Interestingly, all the selection
protocols employed to generate amplification events involve extensive cell killing
and the use of critically narrow ranges of drug concentrations to detect amplifica-
tion events. Any number of studies in my laboratory have shown that pretreatment
with various agents, including inhibitors of DNA synthesis, hypoxia, UV light,
carcinogens, and gamma radiation, can increase the frequency of gene amplifica-
tion in rodent cell lines (CHO and mouse) (see Ref. 20 for references). Charac-
teristically, the degree of cell killing, the time of exposure, and the time following
removal of the perturbing agent when selection pressure is added are all critical
variables. Such treatments markedly increase the frequency of sister chromatid
exchanges [21] and result in metaphase chromosomes with varying degrees of
chromosome breaks, as well as metaphase spreads with extensive extrachromoso-
mal DNA [21,22]. We have interpreted our studies to indicate that such pretreat-
ments alter normal cell cycle progression events and dissociate DNA synthesis and
chromosome condensation from the events of mitosis [22]. Such treatments
facilitate sister chromatid exchange phenomena, are likely to alter replication
progression, and result in chromosome breakage. A major consequence of such
events is cell death. However, recombinational resolution of stalled/altered replica-
tion forks or recombinational repair of broken chromosomes can form the sub-