© 2000 Nature America Inc. • [Link]
com
ANALYSIS
the study of mutagenesis to be easily com- species may require that we be able to com- but with increasing ionic concentrations is
bined with rapid evaluation of develop- bine pharmacological and mutation studies reduced to minutes (data from US Army
ment. Their long history of use in aquatic in isolated tissues. Center for Environmental Health
toxicology has more recently been extended It is true that most tests in transgenic Research). It would be tempting to con-
by the development of molecular markers animals have been conducted at very high clude a great difference in dose response
to detect endocrine disrupters. Given our doses and are likely irrelevant to actual envi- when in fact there is no parity of dose.
awareness of the limitations of these tech- ronmental exposures; typically we expect Interpretation of results from environmen-
niques and our ability to design experi- that environmental doses will be orders of tal exposures must take into account these
ments accordingly, there are tremendous magnitude lower. Fish probably provide one variables in order to establish a reliable
opportunities to understand environmen- of the best opportunities in vertebrates to basis for comparing risk between species
tally induced mutation and make use of study chronic low-dose exposures with good and between locations.
identical transgenes in aquatic models for dose accuracy and control using flow- More than 10 years of experience with
direct comparison with rats, mice, and cells through or renewal designs that can easily various transgenic systems in rodents points
in culture. span generations. This would also be an to some conceptual precautions using a
Emerging issues in environmental toxi- effective way to study acclimation to states transgenic fish mutation system for environ-
cology include (1) low dose and lifetime of either higher or lower sensitivity. We may mental monitoring. The important thing
exposures (2) multigenerational/selection also learn that for such studies the concept isn’t that we have—now in fish—another
effects and acclimation (3) the interaction of point/time comparisons may be insuffi- way to see that mutagens cause mutation. We
between compounds that work additively or cient to discover differences and that it is can, however, investigate the factors in Figure
synergistically, and (4) interactions between necessary to accumulate the mutation data 1 for parallel assessments of mutation, and
synthetic and natural compounds that pro- through time. we can use transgenic fish to address direct
© 2000 Nature America Inc. • [Link]
duce results very different from classical It is also important to consider environ- environmental issues. It all hinges on asking
laboratory exposures. Environmental expo- mental variables associated with differ- biologically relevant questions that the tech-
sures are extremely complex, and use of ences in the water matrix, membrane trans- nology, with whatever limitations it may
aquatic species is probably one of the few port, and bioavailability. One example of have, can answer.
ways that actual mixtures or fractions iso- the influence of water matrix is evident by
lated from the environment can be effec- comparing the chemical half-life (t1/2) of a
1. Amanuma, K., Takeda, H., Amanuma, H. & Aoki, Y.
tively studied. compound in water of increasing ionic Nat. Biotechnol. 18, 62–65 (2000).
Among the numerous technical and conditions. In low-conductivity well water, 2. Environ. Mol. Mutagen. 34, 69–226 (1999).
3. Burkhart, J.G. & Malling, H.V. Environ. Mol.
biological considerations for transgenic the t1/2 for a compound like N-ethyl-N- Mutagen. 22, 1–6 (1993).
animals3 in the detection of mutation, we nitrosourea (ENU) may be several hours, 4. Shane, B.S. et al. Mutat. Res. 377, 1–11 (1997).
need to consider only a couple of them
here. One factor is the relationship between
replication and fixation of mutation.
Experiments with transgenic mice have The use of telomerized cells for
already confirmed an idea put forth by clas-
sical genetics—that increasing replication tissue engineering
rate for a tissue or stage of development
increases the rate at which mutations are
fixed4. Therefore, accurate description of Jerry W. Shay and Woodring E. Wright
events in the exposed animal requires that
we be cautious about the interval between Tissue and organ failure are major health permits the cells to function in a tissue-spe-
treatment and recovery of the vector problems. Surgical repair, artificial prosthe- cific manner. In this issue of Nature
sequence, so that adducts or lesions do not ses, mechanical devices, and both human Biotechnology, Thomas et al. provide data
become mutations in the host animal, but and xenograph organ transplantation con- that supports the feasibility of a first step in
are instead fixed as mutation in the indica- tinue to be important in medical treatment this direction2.
tor bacteria. of certain human diseases1. However, the Normal cells divide a limited number of
A second important consideration (also field of tissue engineering, involving the times before they undergo growth arrest,
partly a replication issue) is the relation- reprogramming of pluripotent stem cells or often referred to as replicative senescence.
ship between samplings and tissues. One of rejuvenation of specific differentiated cells, Telomerase is a cellular ribonucleoprotein
the most salient advantages to using trans- is emerging as a promising alternative. The reverse transcriptase that adds telomeric
genic rodents is the potential to measure eventual goal is to be able to take a patient’s DNA to the ends of chromosomes to com-
differences in mutation frequencies own cells, expand them in a laboratory pensate for the inevitable losses that occur
between tissues. Some species/tissues may environment, genetically engineer them to during replication. The hypothesis that pro-
differentially metabolize a compound to a correct a particular defect, and then rein- gressive telomere shortening could be the
mutagenic form and replicate; others may troduce them into the patient in a form that counting mechanism for replicative senes-
not, and produce few or no apparent muta- cence was recently strengthened by the
tions. Benzo(a)pyrene is one example. demonstration that expression of the cat-
There is also evidence for clusters of Jerry W. Shay (shay@[Link]) and alytic protein component of telomerase
mutants within tissues and the possibility Woodring E. Wright are professors in the (hTERT) could prevent growth arrest and
of rescuing perhaps viable, but altered department of cell biology, University of Texas immortalize human fibroblasts and epithe-
marker DNA from dead cells. Thus, use of Southwestern Medical Center, 5323 Harry lial cells3. Moreover, telomerase is present in
DNA isolated from a whole animals (a Hines Boulevard, Dallas, TX 75390-9039. specialized reproductive cells and almost all
small fish) may mask important data from J.W.S. is a senior scholar, the Ellison Medical cancer cells that appear to divide indefinite-
specific tissues, and comparisons between Foundation, Bethesda, MD. ly4. Although telomerase is present in prolif-
22 NATURE BIOTECHNOLOGY VOL 18 JANUARY 2000 [Link]
© 2000 Nature America Inc. • [Link]
ANALYSIS
erative stem cells of renewal tissues, its level term effects on cancer incidence by immor-
is apparently insufficient to fully maintain talized cells are legitimate, the present
telomere length, since these tissues show results suggest that immortalization or
gradual telomere shortening throughout reversible immortalization of specific cell
life. types that can be thoroughly characterized
Thomas et al. introduced both hTERT before transplantation may have manage-
and SV40 large T-antigen into bovine able risks. In the future, the discovery of
adrenal cells and showed that the modified small molecules or drugs that transiently
cells maintained telomeric DNA2. They activates the normal hTERT promoter or
then transplanted (Fig. 1) these rapidly conditional expression may obviate some of
growing engineered bovine adrenocortical these concerns.
cells into a small (3 mm) polycarbonate Slowing the rate of telomere shortening
cylinder introduced beneath the kidney could have benefits in specific tissues where
capsule of scid (T- and B-cell deficient) telomere-based growth arrest (senescence)
mice that had been adrenalectomized. occurs. The medical implications of this
Whereas the animals without transplanta- technology are profound. Some promising
tion died, animals that received transplants areas of cell engineering include rejuvena-
of cells expressing both hTERT and SV40 tion of hematopoietic stem cells for
large T-antigen survived and produced improving bone marrow transplants or
bovine cortisol to replace the mouse gluco- enhancing general immunity for older
corticoid, corticosterone. This is an impor- patients. Other possibilities include an
© 2000 Nature America Inc. • [Link]
tant result, becasue cortisol secretion is a unlimited supply of skin cells for grafts in
very sensitive measure of cell function, burn patients and for treating ulcerated
since it depends on the formation of a com- lesions that do not heal. The immortaliza-
plete set of steroidogenic enzymes. The tis- tion of chondrocytes or osteoprogenitor
sue formed in the animals was a chimera of cells to treat osteoarthritis or for bone
normal-appearing bovine adrenal cortex grafts, and endothelial cells for the genera-
© Bob Crimi
cells together with mouse endothelial cells. tion of tissue-engineered blood vessels are
The tissue was well vascularized and did not other areas of interest. In the future, we
overgrow the polycarbonate cylinder could try to grow cells in the laboratory for
(whereas a similarly introduced breast can- disorders for which there are currently no
cer cell line did expand outside the cylin- cures—for example, muscle satellite cells
Figure 1. Bovine adrenocortical cells were
der). The proliferation rate in tissues established in culture and transfected with for muscular dystrophy, retinal cells for the
formed by these transplanted bovine adren- SV40 large T-antigen and the catalytic treatment of macular degeneration (a lead-
al cells was low, and there were no indica- protein component of telomerase (hTERT). ing cause of age-related blindness), and
tions of malignant transformation. Approximately 2 × 106 cells were then trans- immune cells for HIV patients. This could
planted into a small cylinder placed beneath
Previously, these investigators had shown the kidney capsule of scid mice that had avoid immune rejections and could slow
that normal bovine adrenal cells and SV40 been adrenalectomized. All mice that down or reverse some of the problems asso-
T-antigen transfected bovine cells were only received cell transplants produced cortisol ciated with these disorders. Alternatively,
transplantable for a short time after cul- instead of the mouse corticosterone, and we could produce proteins from normal
survived despite adrenalectomy.
ture5. Introduction of hTERT greatly human cell cultures to treat the donor
extended the time after culture that trans- patient. Engineered cells may also be useful
plantations were successful. While an would stop dividing, and continue to divide. for in vitro markets, as alternatives to ani-
important question not addressed in this The cells are growing normally, giving rise mal testing, or to produce products for cos-
study is whether hTERT alone could have to normal cells with the normal number of metic applications. However, we will first
accomplished the same result, these experi- chromosomes. Thus, telomerase expression have to develop safety and efficacy stan-
ments dramatically showed that an and maintenance of telomere integrity does dards, quality and control assurances, and
endocrine tissue could be derived from a not bypass cell cycle-induced checkpoint preclinical and clinical evaluations. The
previously cultured somatic cell type that controls and does not lead to genomic insta- present results are a first step in document-
expressed both T-antigen and hTERT. bility. However, anchorage-independent ing that the introduction or activation of
Another important result in these stud- growth and xenograph tumors can be telomerase in normal cells is likely to have
ies is that the combined introduction of obtained if T-antigen is introduced into many applications and a major impact for
viral oncoproteins together with telomerase normal human cells followed several the future of medicine.
did not result in cancer progression. The months later by expressing both H-ras and
expression of telomerase in almost all hTERT (ref. 8). The experiments by Thomas
1. Persidis, A. Nat. Biotechnol. 17, 508–510 (1999).
malignancies suggests that overcoming the et al.2 are important because they demon- 2. Thomas, M., Yang, L. & Hornsby, P.J. Nat.
proliferative limits imposed by telomere strate that in some cell types a cancer phe- Biotechnol. 18,39–42 (2000).
3. Bodnar, A.G. et al. Science 279, 349–352 (1998).
shortening represents a key step in oncogen- notype is not obtained, even after many 4. Kim, N.-W. et al. Science 266, 2011–2015
esis4. Initial concerns that the introduction normal cell cycle checkpoint functions have (1994).
of telomerase alone into normal cells might been abrogated. It would be of interest to 5. Thomas, M., Northrup, S.R. & Hornsby, P.J. Nat.
Med. 3, 978–983 (1997).
actually transform them have not been know if the introduction of H-ras into these 6. Morales, C.P. et al. Nat. Genet. 21, 115–118
borne out6,7. The cells with introduced telomerized, T-antigen expressing bovine (1999).
7. Jiang, X.-R. et al. Nat. Genet. 21, 111–114
telomerase have extended the length of their adrenal cells prevents successful transplan- (1999).
telomeres, have continued to divide for over tation or produces premalignant or malig- 8. Hahn, W.C. et al. Nature 291, 464–468 (1999).
300 generations past the time they normally nant changes. While concerns over long-
NATURE BIOTECHNOLOGY VOL 18 JANUARY 2000 [Link] 23
