Download DNA microarray technology: insights for oral and maxillofacial

British Journal of Oral and Maxillofacial Surgery (2004) 42, 542—545
DNA microarray technology: insights for oral and
maxillofacial surgeons
J.R. Bowdena,∗, P.A. Brennanb
a
Department of Oral and Maxillofacial Surgery, Southampton General Hospital, Tremona Road,
Southampton SO16 6YD, UK
b Queen Alexandra Hospital, Cosham, Portsmouth PO6 3LY, UK
Accepted 9 August 2004
Available online 22 September 2004
KEYWORDS
DNA microarray;
Gene chip;
Review
Summary Since the discovery of DNA and the recent sequencing of the entire
human genome, there have been great advances in our understanding of the genetic
basis of many diseases, including cancer.
An exciting recent development is DNA microarray technology. This technique allows many genes to be studied in the same experiment rather than one gene at a
time. It can therefore provide large amounts of data much more quickly than was
previously possible. This has great implications for diseases such as cancer, which
often show genetic damage in many parts of the genome. DNA microarray technology has now been used in the investigation of many tumours including melanoma,
breast cancer, and lymphoma, as well as in the understanding of the genetic basis
of metabolic diseases. However, it seems that our specialty knows little about the
technique and its possible clinical applications. We give here a simple introduction
to the technology and its likely role in the future management of oral cancer.
© 2004 The British Association of Oral and Maxillofacial Surgeons. Published by
Elsevier Ltd. All rights reserved.
Introduction
It is over 50 years since Watson and Crick established the molecular structure of deoxyribonucleic
acid (DNA) and its potential genetic applications.1,2
DNA is a double helical structure, each strand composed of a sugar, a phosphate and a base (Fig. 1).
* Corresponding author. Tel.: +44 2380 796096;
fax: +44 2380 798640.
E-mail address: [email protected] (J.R. Bowden).
The sugar (␤-d-2-deoxyribose) is linked by phosphate groups to form the unchanging backbone
structure of DNA. The bases form the variable component, consisting of either a purine (adenine, A
or guanine, G) or a pyrimidine (thymidine, T or cytosine, C). The bases in each strand of the double
helix are linked to form complementary pairs; adenine pairs only with thymidine and guanine pairs
only with cytosine (Fig. 1). It is this exact pairing
of bases that gives DNA its unique ability to act as
a template in replication.
0266-4356/$ — see front matter © 2004 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjoms.2004.08.009
DNA microarray technology
543
or RNA sequences in one experiment. The process
is the reverse of Southern blotting, the probe being
placed on an immobile surface and then exposed
to the free nucleic acid to be analysed, known as
the target (Fig. 2). The application of the chemical
process is not new; it is the scale on which it can be
used that has made it such an astounding success.6
Manufacture of microarrays
Figure 1. The double helical structure of DNA showing
how the bases, sugar, and phosphate connect with each
other.
An understanding of the chemical structure is essential when considering all the applications of DNA
technology that have been described subsequently.
Since the pioneering work of Watson and Crick,
biotechnological research has increased exponentially and sequencing of the entire human genome
is now complete.3 As a consequence, public interest
has increased rapidly, and the scrutiny of the media has accelerated the rush for further advances
in medical and scientific research.
A recently described technique of DNA microarray is a further exciting advance that will have
important clinical applications, particularly in relation to cancer. However, most oral and maxillofacial
surgeons know little about this new technology.
Here, we review the basic scientific aspects of DNA
microarrays and the implications for our specialty.
Several different microarray techniques, devices,
and instrument systems have been developed and
are now commercially available for producing DNA
microarrays.7 This is done by depositing the probes
and immobilising them on a substrate, usually made
of glass, silicone, or nylon. The actual manufacture
of microarrays is either by photolithography or by
a ‘‘spotting’’ process. The photolithography technique borrows technology from the semiconductor
computer chip industry to allow parallel synthesis
of large numbers of oligonucleotides on a solid surface, so that expression of up to 400,000 distinct
sequences can be produced on one slide.5 A spotting technique is akin to the piezo-electric process
of the ink-jet, in that probes are blown on to the
substrate, under precise robotic positional control.
Terminology
Two nomenclatures have been used for hybridisation techniques, which can lead to some confusion.
When applied to a microarray it parallels a reverse
dot blot in that a probe is immobilised and tethered
and the free nucleic acid is the target; in contrast
to a conventional Southern blot in which the target
is immobilised and the probe is free.
Basic science
Clinical applications of DNA microarrays
What is a DNA microarray?
Many research groups have attempted to use DNA
microarrays to solve diverse clinical problems. An
enormous amount of data can be generated in microarray analysis and it is necessary to sort out what
is meaningful in this wealth of information. It has
indeed been likened to going on a ‘‘fishing expedition’’.8
Several clinical areas and applications are worthy of special consideration.
The ability to label nucleic acid molecules and then
use them to interrogate other nucleic acids attached to a solid support was first described over
25 years ago.4 This technique, eponymously named
Southern blotting, uses a DNA probe labelled with
either a radioisotope or a fluorescent tag. The
probe is applied to the fragment of DNA or RNA to
be studied and by the rules of base pairing (A to T, C
to G) will ‘‘stick’’ to its complementary sequence.
Indeed it has been described as the first type of
array.5 Advances in technology have made it possible to miniaturise methods of probe detection for
DNA and allow detection of several thousand DNA
Expression profiling
The phenotype of any organism is established by
the expression of many genes. DNA microarrays allow for the study of mRNA, which has been ex-
544
J.R. Bowden, P.A. Brennan
Figure 2. Complementary DNA from tumour cells is labelled with a fluorescent dye and applied to a microarray. The
different molecules of DNA attach to their corresponding genes. The same procedure is done with a control group of
cells with a different colour of fluorescent dye. A laser scans the microarray and analyses the intensity of the different
colours to give information about each gene.
tracted from fresh tissue and reverse transcribed.
This gives an indication of the active genes; and
leads to the production of a gene expression profile or signature.9,10 The identification of multiple
genes has the potential to lead to the identification of metabolic pathways and pathogenetic mechanisms, new indicators of disease progression, and
new drug targets.11 In individual patients, the identification of certain expression profiles can lead to
a more accurate diagnosis and specifically to individually tailored treatment and prognosis. It may be
that the use of DNA microarrays to provide a genetic
signature will become part of the routine investigation in conjunction with history, examination,
and histological examination of any patient with
malignancy. Indeed, expression profiles of acute
leukaemia,12 B-cell lymphoma,13 melanoma,14 and
breast carcinoma15 have led to advances in methods
of staging and classifying these diseases dependent
on the expression of genes in individual tumours or
cell populations. Of particular note was the study
on B-cell lymphoma,13 as within this group of cancers two distinct gene ‘‘fingerprints’’ were found,
reflecting different stages of B-cell development.
The two groups had considerably different prognoses, 80% surviving 5-years in one group and 40%
in the other. It is interesting to note that patients
with melanoma could be subdivided into two distinct groups based on their gene expression profiles,
even though there were no obvious pathological differences between the tumours.
Genotyping
Work on the human genome project has led to
the identification of numerous single nucleotide
polymorphisms (SNP) as genetic markers. It is
now possible to identify mutations that underlie
susceptibility to specific diseases. This could lead
to the recognition of an individual patient’s risk
of developing a specific disease such as diabetes,
asthma, and coronary heart disease.
Possible role of DNA microarray technology
in oral and maxillofacial surgery
For the practising oral and maxillofacial surgeon the
sequencing of the human genome and DNA microarray technology may seem of little relevance. However, in the future this technology promises to give
a better understanding and insight into biological
processes.
The development and progression of squamous
cell carcinoma in the head and neck is a complex
process and involves progressive genetic damage,
loss of heterogeneity and the inactivation of tumour
suppressor genes.16,17 Furthermore, even though
tissue margins close to resected tumours may look
microscopically normal, there is increasing evidence that genetic damage may be widespread in
oral epithelial cells, which can explain synchronous
tumours.18 DNA microarray technology will be use-
DNA microarray technology
ful in assessing the degree of genetic damage in
both the primary tumour and the surrounding mucosa, which could alert clinicians to the probability
of tumour recurrence. In the foreseeable future, it
is likely that all tumours will be analysed by DNA
microarray technology, and it may allow targeting
of individual treatment by predicting the sensitivity of each tumour to chemotherapy.19,20 It may also
be useful in predicting those cases of dysplasia that
will undergo malignant transformation. In this case
close follow up or early treatment of the dysplastic
mucosa may prevent the later development of malignancy. We envisage that it may also have benefits
in assessing the malignant potential of oral lichen
planus. It will lead to a greater understanding of the
process of development by identifying which sets of
genes are responsible for coordination and regulation of growth of the craniofacial skeleton, with implications for establishing the cause and potential
treatment of developmental anomalies.21
We are interested in how tumours develop a new
blood supply (angiogenesis) and how oral cancer
cells facilitate their own invasion into underlying
connective tissue. We are now using specific commercially available DNA arrays that are designed to
analyse a wide range of genes involved in the angiogenic process. Rather than assessing one or two
aspects of angiogenesis, we are able to look at 96
different genes in one array, including those responsible for vascular endothelial growth factor (VEGF),
fibroblast growth factor (FGF), and cyclo-oxygenase
(COX) enzymes. It is possible that this type of DNA
microarray will be useful in predicting the likelihood of metastasis in oral cancer. Although there
are many studies that have found correlations between lymph node metastases and various angiogenic factors, there is as yet no single investigation
that can readily assess all of these factors in one
experiment.
In conclusion, DNA microarray technology is an
exciting, rapidly expanding field but at present little is known about it in our specialty. We anticipate
that it will enable both a greater understanding of
the biology of oral cancer, and help in the clinical
management of this disease.
Acknowledgement
We thank the British Association of Oral and Maxillofacial Surgeons for the award of an endowment
grant.
545
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