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Molecular strategies are
currently being either used or sought for brain tumors, stroke,
neurodegenerative diseases, vascular malformations, spinal degenerative
diseases, and congenital malformations of the central nervous system.
Many of these patients have been treated for neurological diseases for
which conventional medical therapies have been of limited utility. These
methods include commonly used techniques such as advanced cytogenetics,
differential display, microarray technology, molecular cell imaging,
yeast two-hybrid assays, gene therapy, and stem cell utilization.
Of late, there is increasing interest in use of molecular strategies in
brain tumors. The neuro-oncologist of the next century will be a
molecular biologist. This may result in brain mind manipulation including
the manipulation of learning, memory and several types of emotions.
MOLECULAR BIOLOGY:
The cancer viruses were discovered in 1911, but largely ignored for several
decades. They came into focus again in 1950s.The central task was to
isolate them and prepare vaccines for immunization. However it became
problematic because viruses seemed to play an etiological role in just a
handful of human cancers.
In 1960s somatic cell fusion led to systemic mapping of somatic genomes.
In 1969 Huebner and Todaro proposed the concept that in multicellular
organisms, genes responsible for regulation of normal development could
go awry. The results would be the misregulated growth typical of cancer.
The term oncogene was introduced to describe such genes, and the concept
become known as the oncogene hypothesis. This theory defines a genetic
basis for cancer; it links normal cellular functions of growth and
differentiation (mediated by proto-oncogenes) with neoplastic
transformation (where proto-oncogenes become oncogenes) and it provides
the unifying theory whereby the ability of carcinogenes and other genetic
disturbances, including the insertion of cancer viruses to contribute to
oncogenesis may be explained.
Investigations:
Assays are generally divided into two types: structural assays and
functional.
Structural assays determine the configuration of building blocks of large
molecules, eg, the arrangement of genes on a chromosome, & the
sequence of nucleotides in the DNA.
Functional assays determine how things act eg, which genes are
transcribed in particular cells, whether cells are normal or malignant,
how well a protein or its mutant counterpart can perform a particular
task.
Recombinant DNA technology encompasses restriction enzymes, probes,
Southern blotting, gene cloning, and sequencing, and polymerase chain
reaction.
Restriction enzymes:
These enzymes typically recognize four to six base pair sequences in the
DNA and break the double stranded molecule in or near that region. These
enzymes are named for the bacterial strain of origin or order of
discovery. Some 300 restriction strain enzymes have been isolated,
recognizing more than 100 different sites.
In 1987, Seizinger studied restriction fragment length polymorphisms
(RFLPs) in normal and tumor cells. These polymorphisms are stably
inherited variations in chromosome structure without pleomorphic
expression, revealed by a different pattern of restriction enzyme
digestion. This means that with the same restriction enzyme, the DNA
double helix may be cut on different places considering the maternal and
paternal counterparts of one chromosome. By themselves RFLPs do not imply
loss of function or a tendency towards neoplasm, but rather variants of
DNA restriction enzymes recognition site locations leading to different
lengths of a given sequence, after digestion of restriction enzymes.
Restriction mapping:
Once the length of the DNA can be partitioned into a finite number of
consistent fragments (due to the action of specific restriction enzymes)
the study of the DNA is clearly facilitated. The number of recognition
sequence sites cleaved by the enzyme is directly proportional to the time
of digestion. The rate at which these fragments move on in an electric
field is indirectly proportional to their size, with smaller fragments
moving more rapidly than larger ones. By comparing fragment sizes it is
possible to construct detailed maps. If the size of DNA increases, it
becomes more difficult to order restriction fragments: in these cases,
smaller, more manageable pieces of DNA are generated and multiplied by
molecular cloning for further detailed investigation.
DNA Cloning:
The objective of cloning DNA is to produce large numbers of identical
copies of particular DNA fragments. This goal is most easily accomplished
by taking advantage of prokaryotic systems with their capacity of rapid
reproduction, limited only by the availability of nutrients. There are
essentially two methods of producing large copy numbers of foreign DNA
fragments in bacteria. They are, use of plasmids or use of bacteriophages
as cloning vectors.
Probes:
It is possible to isolate total DNA from complex eukaryotes such as human
cells and to clone the entire genome. Such a set of clones is called a
genomic library. To identify a specific region of interest, radioactive
sequences of DNA or RNA (probes) complementary to the desired gene can be
used. The original probes are prepared from RNA tRNA, rRNA or Mrna. The
mRNA encoding for the protein of our interest will be converted to DNA (
cDNA for copy DNA ) by using the reverse transcriptase enzyme of a
retrovirus. New cDNA probes can be characterized by using them to
reisolate the mRNA from which they were made, which in turn is translated
into the protein of interest. As such, specific genes responsible for the
expression of specific proteins can be identified. A bank of probes has
been developed that reveals specific genes on chromosomes and also
reveals RFLPs.
Southern blotting:
In 1975, EM Southern developed one of the most important techniques in
molecular biology, a procedure enabling the identification of specific
fragments of DNA out of many thousands generated from the human genome
through the use of restriction enzymes, which cleave the DNA at specific
sites. After separation of the fragments by size, a probe is used to
discriminate among them for the sequence of interest. Fragments are
separated according to size. The DNA from the gel is denatured with a
basic solution to give single strands. The single stranded fragments are
transferred to a membrane. Thus a replica or blot of the DNA fragments on
the original gel is made on the filter. Once the replica is obtained,
sizes of specific DNA fragments can be determined by hybridizing the
filter with a radioactive probe in solution. The probe binds to any
complimentary DNA and can be visualized by autoradiography. By comparing
the number sizes and amounts of radioactivity in various DNA fragments,
an estimate of relative gene abundance can be deduced. In other words the
presence or absence of particular oncogenes can be analyzed.
In practice, when a patient is heterozygous for a specific RFLP on
chromosome 22 for example this implies that the normal cell's two copies
of chromosome 22 ( homologous ) have a different DNA structure in the
region being examined. The restriction enzyme will cleave the two copies
of DNA at another place. So, after hybridization of this sequence with a
sequence specific probe, two bands on southern blotting specific for each
locus will appear. This allows one to detect loss of one copy of the two
loci (for example oncogenes) if the tumor has lost its heterogeneity for
that locus. This is known as loss of heterozygosity (LOH) studies.
DNA Sequencing:
This technique developed in 1975, isolates a single stranded unlabelled
DNA fragment and a short end labelled complimentary primer is attached by
its 5' end to the 3' end of the longer fragment. The Maxam - Gilbert
method utilizes specific chemical cleavages to differentiate among the
DNA bases: a double stranded DNA fragment is labeled with radioactive
phosphate at both 3' and both 5' ends by a 3' or 5' kinase. These
techniques have illuminated the primary gene structure, which in
eukaryotes includes the sequence of both coding regions (axons) and
intervening sequences (intones), as well as the number and arrangement of
these regions.
Polymerase chain reaction:
The polymerase chain reaction takes advantage of the ability of a small
piece of DNA to find and bind to a target sequence on another stretch of
DNA. It then uses an enzyme called polymerase to make a new piece of DNA
corresponding to the region adjacent to the target. In the original form
of PCR, the researchers had to know the sequence of bases on either side
of the target DNA before making short stretches of DNA. These primers are
complimentary to the sequences. Heat separates the two strands of the
DNA, the short primers find their complementary sequences and bind to
them. A polymerase and nucleotide are added. This enzyme joins
nucleotides the building blocks of DNA, into a new strand of DNA,
attached to the promer. Each such cycle of new heating, cooling and
polymerization doubles the number of copies of target DNA. The ends of
all these copies are the same. Thus the amplified sequence can be
purified by running the DNA through an electrophoretic gel. This sorts
out the DNA by size; all copies of the target, because they are all of
the same length, will lie in a neat band on the gel. We can easily become
more than a billion copies of our target DNA region, starting with one
single strand DNA double helix, i.e starting from just one cell.
Oncogenes and
carcinogenesis:
Cellular Proto-oncogenes (normal growth and differentiation genes)
oncogene products (i.e. proteins) are expected to control cell growth.
Various onc proteins have been localized to the nucleus, cytoplasm and
membranes as well as to the cell surface. The ubiquitous distribution of
proto-oncogenes throughout the vertebra phylum suggests a fundamental
role for these genes.
The oncogene hypothesis proposes that normal genes involved in
development or differentiation may be altered in such a way that their
products transform the cell to neoplastic growth.
Potential mechanisms are numerous:
DNA Changes -
a) Rearrangements b) Insertion / deletion c) Point mutation (s) d)
Amplification - increased copy number.
RNA Changes -
a) Strong promoter insertion > increased copy number b) Enhancer >
increased copy number c) Processing Mutation d) Fusion Message.
Protein changes -
a) Too much/too little b) Altered function c) Altered Stability
The inheritance of cancer appears actually to be the inheritance of a
predisposition to cancer. Every cell in the affected area does not itself
undergo malignant transformation. Two different oncogenes can act in a
complimentary fashion to convert normal cells to a tumorigenic state. Oncogenes,
and tumor suppressors may also influence both early and late stages of
tumor progression. Oncogenes are considered to act in a dominant manner
if expression of one transforming allele contributes to a neoplastic
phenotype. There is evidence for another class of genes whose expression
inhibits transformation. Loss of such genes may lead to cancer. The
existence of cancer suppressor genes was suggested first by somatic cell
hybridization experiments. Tumorigenesis is a result of inactivation
(deletion, mutation) of both copies of suppressor gene on the two
homologous chromosomes in a tumor cell. These two somatic mutations may
happen during a life span or there may be an inherited mutant allele on
one mutation on the corresponding allele in this patient is enough to
induce cancer.
GENE THERAPY:
Gene therapy may be defined as the transfer of genetic material into a
patient's cells for therapy.
Gene therapy is a novel approach to treating diseases based on modifying
the expression of a person's genes toward a therapeutic goal. Gene
therapy is most often been discussed in the context of treating lethal
and disabling diseases although it also has a potential for disease
prevention. With years of clinical trials, there is some recent evidence
that gene therapy may be efficacious in the treatment of certain single
gene deficiency diseases. Nevertheless, gene therapy remains a highly
experimental collection of technologies whose full potential is yet to be
realized.
Genes we inherit from our parents influence virtually every human
disease. Several years ago, an international effort was launched to
identify every single human gene. This effort, called the Human Genome
Project, is largely completed and the data suggests that each individual
human being may have on the order of 30,000 genes. Unfortunately, some of
this genetic heterogeneity leads to the development of disease.
Inheritance of a single gene is the cause for inheritance of genetic
diseases.
The premise of gene therapy is based on correcting disease at the level
of DNA molecules and thus compensating for the abnormal genes. There are
essentially two forms of gene therapy.
Somatic gene therapy involves the manipulation of gene
expression in cells so as to be corrective for the patient, but this
correction is not inherited by the next generation. This is the type of
gene therapy that is currently being investigated.
Germline gene therapy involves the genetic modification of
germ cells that will pass the selected change on to the next generation.
Research on germline intervention is strictly limited to animal model
systems, and there is no intent to pursue this type of approach in humans
at any time in the near future; this is because of significant technical
and ethical challenges.
Delivery:
"Drug delivery," where the gene is a drug, is particularly
challenging. If genes are optimally delivered, they can persist for the
life of the cell and potentially lead to a cure.
Vectors are gene delivery vehicles, which encapsulate therapeutic
genes for delivery to cells. Many of the vectors currently in use are
based on attenuated or modified versions of viruses. Our challenge
is to remove the disease-causing components of the virus and insert
recombinant genes that will be therapeutic to the patient. The modified
viruses cannot replicate in the patient, but do retain the ability to
efficiently deliver genetic material. The purpose of these vectors is to
track down tumor cells and destroy them by delivering a payload of genes.
These genes make proteins that either kill the cells directly or make
them susceptible to chemotherapy. A variety of different strategies have
now been pursued in clinical trials.
Another strategy is based on non-viral vectors in which complexes
of DNA, proteins, or lipids are constructed as particles capable of
efficiently transferring genes.
Trials began in 1990, using an ex vivo strategy. In this approach, the
patient cells were harvested and cultivated in the laboratory and then
incubated with vectors to introduce the therapeutic genes. The cells were
then harvested and transplanted back into the patient from whom they were
derived. The field moved quickly into more practical approaches for
delivering genes based on in vivo gene therapy in which the virus is
directly administered to the patients.
This enabling technology is being used to develop treatments for such
diverse diseases as cancer, cardiovascular disease, and AIDS. In fact,
the most common disease target represented in the approved clinical
research protocols is cancer; this accounts for approximately 60% of all
the trials being conducted.
Current approaches for gene therapy of CNS disorders include the
following:
1)Gene replacement with a single normal allele to correct the inherited
global neurodegenerative disorders, such as enzyme deficiencies.
2)Brain repair to restore the function of a particular subset of
cells that were lost because of a neurodegenerative process and
stroke.
3)Gene therapy for brain tumors:
Most malignant brain tumors are fatal, and surgery and radiation may add
only a few months to the patient's life. The prognosis for these tumors
remains essentially unchanged despite significant advancements in
neuro-oncology and radiation therapy. Hence the search for newer
therapies continues. It is hoped that gene therapy can one day be used as
an additional treatment for malignant brain tumors.
Many therapeutic transgenes have shown efficiency in experimental models,
including generation of toxic compounds, enzymatic activation of
prodrugs, expression of tumor suppressor or apoptotic proteins,
inhibition of angiogenesis and enhancement of immune responses to tumor
antigens.
Herpes virus is a good platform because it enters human cells
readily, is big enough to carry numerous transgenes, and easily threads
its genetic material into the host's nuclear DNA; retrovirus, adenovirus
and adeno-associated virus have also been in use in brain tumors.
A variety of strategies have now been used in clinical trials:
1) Gene directed enzyme prodrug (suicide gene) therapy (GDEPT).
2) Therapy to boost the immunity against cancer cells.
3) Transfer of potentially therapeutic genes, such as tumor suppressor
genes into cancer cells.
4) Oncolytic virus therapy.
5) Antisense therapy.
GDEPT strategy is most extensively used. In the laboratory, scientists
change the virus by removing the gene for making the virus reproduce and
replacing it with a gene that will make the enzyme, thymidine kinase. The
altered virus is injected into the brain tumor's center with the use
stereotaxy to determine the precise location for the injection. Once in
the tumor, the adenovirus makes thymidine kinase. When exposed to the
drug ganciclovir, thymidine kinase causes the drug to become a toxin.
Ganciclovir has no effect on healthy human cells.
Presently high grade primary brain tumors (glioblastoma and astrocytoma),
recurrent and those in the so-called critical regions (brainstem, speech
area, etc), and childhood tumors are selected for gene therapy in various
trials. Ideally, the tumors are in the volume range of 50-100 cm in a CT
scan.
After surgically removing as much of the patient's tumor as possible,
neurosurgeons could apply gene therapy to reach any remaining, unseen
tumor cells before closing the skull and ending surgery. The gene therapy
virus would "infect" mainly the cancerous cells because the
virus targets dividing cells.
Although patients have not been cured, gene therapy not only gives
neurosurgeons a potential tool to control tumor growth, but also could
make radiation therapy more effective.
A lot depends on the genetics of the individual tumor, some tumor cells
will not. For that reason, a combination of genes will probably prove
most useful for therapy. A mixture would also be useful when tumor cells
are resistant to a particular tumor suppressor gene.
The ongoing trials, it is hoped, will offer better cure to some of the
most devastating malignancies affecting both children and adults and add
to the surgeon's armamentarium.
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