Medical Molecular Biology
Transcript Medical Molecular Biology
Medical Molecular Biology
As to diseases, make a habit of two things
– to help, or at least to do no harm.
Epidemics, in Hippocrates, translated by W.H.S.
Jones (1923), Vol. I, 165
• Discoveries in molecular biology have led
to many advances in medicine.
• Increased understanding of the nature of
• Development of treatment strategies.
17.2 Molecular biology of
“If thou examinest a man having
tumors on his breast… There is no
From the Edwin Smith Papyrus, an ancient
Egyptian medical manuscript that dates back to
approximately 1600 B.C.
Who will develop cancer in their lifetime in
• One half of all men.
• One third of all women.
Cancer is a multistep disease
• Not just one disease but a group of genetically
• Each tumor can have its own “genetic
• Accumulation of many (~4 to 8) genetic
changes over the course of years.
• Gene mutations that increase the risk for
developing cancer can be inherited or
Three major changes that occur when a
cell becomes cancerous:
Genetic changes associated with
• Gain of function
– Inappropriate activation of oncogenes
• Loss of function
– Inactivation of tumor suppressor genes
Activation of proto-oncogenes and
• Oncogenes are genes whose products have
the ability to cause malignant transformation of
• Originally identified as the “transforming genes”
carried by some DNA and RNA tumor viruses.
• Proto-oncogenes are cellular genes with the
potential to give rise to oncogenes.
• Oncogenes are referred to by three-letter
codes, reflecting the retrovirus from which they
• Designated by “v-” to indicate their original
identification in retroviruses.
– e.g. v-src, v-myc
• “c-” signifies the cellular proto-oncogene
Inappropriate activation of protooncogenes may be due to:
• Qualitative changes
– Mutations in the coding sequence
• Quantitative changes
– Inappropriate regulation of expression
Proto-oncogenes can be classified into
many different groups:
• Secreted growth factors.
• Cytoplasmic proteins such as serine kinases
and tyrosine kinases.
• Surface and membrane-associated proteins
involved in signal transduction.
• Transcription factors.
v-src tyrosine kinase
• The first oncogene discovered.
• v-Src differs from c-Src by substitutions
of sequences at the C-terminus.
• Remains in the “open” or active
conformation and constitutively
phosphorylates target proteins.
How cancer cells metastasize:
the role of Src
• A primary role of c-Src is to regulate cell
adhesion, invasion, and motility.
• All of these basic cellular processes are
deregulated during tumor progression
c-myc transcription factor
• c-myc is a central “oncogenic switch” that
regulates a diversity of cellular functions
through altering gene expression.
• Encodes a helix-loop-helix transcription factor
that dimerizes with Max.
• c-myc overexpression is often correlated with
highly aggressive tumors.
Inactivation of tumor suppressor genes
• Tumor suppressor genes normally inhibit
• Cancer arises when they are not
Two well-characterized tumor suppressor
• Retinoblastoma protein (pRB)
• p53 protein
Knudson’s two-hit hypothesis and
• Cancer arises when there are two
independent mutations or “hits” that lead
to loss of function of both tumor
suppressor alleles at a locus.
• If loss of one allele inherited through the
germline, an individual is said to have a
“genetic predisposition” to cancer.
Retinoblastoma protein: the cell cycle
• First tumor suppressor protein gene to be
• Deletion of the gene is linked to
retinoblastoma, a childhood hereditary
cancer syndrome resulting in tumors of
• The cell cycle is driven by the
coordinated activation of cyclindependent kinases (CDKs).
• Hypophosphorylated pRB binds the E2F
transcription factor complex and prevents
it from binding to target genes.
• Phosphorylation inactivates pRB.
• Inactivated pRB releases the E2F
• E2F activates expression of genes
needed for S phase.
• What occurs during S phase?
• CDK2 catalytic activity in acute
lymphoblastic leukemia cells keeps pRB
in a phosphorylated state.
• pRB is thus effectively “absent.”
p53: the “guardian of the genome”
• Regulates multiple components of the
DNA damage control system in response
to cellular stress signals.
• In normal cells there are low levels of p53
because p53 is targeted for proteasomal
• p53 is activated in response to cellular
stress, such as UV irradiation.
If DNA damage occurs early in G1:
• p53 regulates the expression of genes such as
the CDK inhibitor p21.
• Cell cycle arrest.
• DNA repair.
If DNA damage occurs later in the cell
• p53 promotes apoptosis.
Role of p53 in cancer
• 80% of all human cancers show either:
– Deletion of both alleles leading to the
absence of the p53 protein.
– A missense point mutation in one allele and
production of a dominant negative protein.
• 60% of all lung cancers in cigarette
smokers show inactivating mutations in
The discovery of p53
• Researchers first concluded that p53 was an
• A p53 cDNA clone derived from “normal” liver
cells turned out to be a mutant form of p53 with
• Wild-type p53 was shown not to have
Inappropriate expression of microRNAs
• Upregulation or downregulation of some
clusters of miRNAs is associated with a
number of types of cancer.
• The pattern of miRNA expression varies
dramatically across tumor types.
• OncomiRs – oncogenic microRNAs.
• Overexpression of the mir-17-19b miRNA gene
cluster accelerates c-myc-induced B cell
• Burkitt’s lymphoma
• Acute promyelocytic leukemia
• Chronic myelogenous leukemia
• In acute promyelocytic leukemia, a
chromosomal translocation brings together
PML and RAR genes to form a fusion
• PML-RAR recruits HDAC and inhibits the
transcription of retinoic acid-responsive
target genes and p53 function.
• In chronic myelogenous leukemia the
BCR-ABL fusion protein has unregulated
tyrosine kinase activity.
• When the drug imatinib occupies the
kinase pocket instead of ATP, the action of
BCR-ABL is inhibited.
Viruses and cancer
• Tumor viruses are of two distinct types
– Those with DNA genomes.
– Those with RNA genomes.
DNA tumor viruses
• Possible outcomes of DNA tumor virus
– A productive infection in “permissive” cells.
– Transformation of “nonpermissive” cells.
• DNA tumor virus transformation is the
result of interaction between viral-encoded
proteins and the host cell proteins.
• Inhibition of normal tumor suppressor
function of host cell proteins.
DNA tumor viruses and human cancers
• Hepatitis B virus: liver cancer.
• Human papilloma virus: penile, uterine,
and cervical cancer
• Epstein-Barr virus: Burkitt’s lymphoma and
nasophyrngeal cancer, B cell lymphoma,
and Hodgkin’s lymphoma.
Human papilloma virus (HPV)
and cervical cancer
• Low risk HPVs cause genital warts.
• High risk HPVs can cause lesions that progess
to invasive squamous cell carcinoma.
• High risk HPVs also cause a vary rare skin
condition called epidermodysplasia
• Viral protein E6 inactivates p53.
• E6 also activates transcription of the gene
encoding the reverse transcriptase subunit
• Viral protein E7 inactivates pRB.
RNA tumor viruses (retroviruses)
• Most human cancers are probably not the
result of retroviral infection.
• Increased risk of cancer associated with
HIV-1 and HTLV-1.
Retroviruses can transform cells by either of
two main mechanisms
• Introduction of an oncogene.
• Promoter/enhancer insertion.
Retroviral introduction of
• Many retroviruses lose part of their genome
• The protein encoded by the oncogene is often
part of a fusion protein with other virally-encoded
amino acids attached.
• The virus may require a helper virus to replicate.
• Chemical carcinogens or their metabolic
products can either directly or indirectly
affect gene expression:
– Genotoxic effects
– Nongenotoxic effects
• Benzo(a)pyrene in cigarette smoke
– The formation of DNA adducts that interfere
with replication and transcription.
– Chromosomal rearrangements.
• Benzo(a)pyrene in cigarette smoke also
promotes tumor formation by:
– Altering arylhydrocarbon receptor-mediated
signal transduction pathways important for
cell cycle control.
– Upregulated genes include members of the
CYP family of enzymes that convert
procarcinogens to carcinogens.
17.3 Gene therapy
Somatic cell gene therapy
• A malfunctioning gene is replaced or
compensated by a properly functioning gene in
somatic cells of a patient.
• Not heritable.
• Treatment only affects the individual patient.
• Germline gene therapy would entail
genetic modification of gametes or
embryos, such that changes are passed
on to the next generation.
• There are many ethical considerations and
such therapy has not been attempted.
Over 900 somatic cell gene therapy clinical
protocols in progress:
• Approximately two-thirds for the treatment of
various forms of cancer.
• Approximately one-third for the treatment of
cardiovascular disease, infectious disease, and
inherited autosomal recessive disorders.
Cancer gene therapy:
a “magic bullet”?
• Licensing and marketing of Gendicine in China.
• Recombinant adenovirus vector carrying the
p53 tumor suppressor gene.
• In one study, 64% of patient’s tumors showed
• Goal: knockdown of disease-related gene
expression in humans.
• First clinical trial: treatment of macular
• Animal model: RNAi can lower
cholesterol levels in mice.
Vectors for somatic cell
• Two main strategies for somatic cell gene
– In vivo
– Ex vivo
• Gene therapy has proved disappointing
time after time.
• Current lack of success due to difficulties
with gene transfer vectors:
– Lack of expression or inappropriate
– Immune response.
– Activation of an oncogene.
• The majority of gene therapy trials use
• Use of liposomes and naked DNA has
Three main viral vectors for somatic cell
• Retrovirus vectors
• Adenovirus vectors
• Adeno-associated virus vectors
Retroviral-mediated gene transfer: how
to make a “safe vector”
Three main steps:
1. Construct provirus carrying selected therapeutic
gene and psi () sequence required for inclusion
of RNA in a viral particle.
2. Insert into packaging cell line the contains a
helper provirus that lacks the sequence.
3. Incubate “safe vectors” with target cells.
The safe vectors contain the reverse
transcriptase, the therapeutic RNA, and other
viral proteins, but do not contain any genomic
Since no viral proteins are encoded by the
therapeutic provirus, the virus cannot
reproduce or form infectious particles.
• Originally thought the retroviral vectors
inserted randomly into the host genome.
• In fact, retroviral vectors preferentially
insert near active genes.
The first gene therapy fatality
• Prior to September 17, 1999, the general
consensus was that somatic cell gene
therapy for the purpose of treating a
serious disease was an ethical
Treatment of Jesse Gelsinger for partial
ornithine transcarbamylase (OTC) deficiency.
Injection of recombinant adenovirus into portal
Four days later, Jesse died from systemic
inflammatory response to viral proteins with
multiple organ failure.
Enhancement genetic engineering
• A gene is inserted to “improve” or alter a
characteristic or a complex trait that
depends on many genes plus extensive
interaction with the environment.
• e.g. “Schwarzenegger mice”
Gene therapy for inherited
• Gene therapy has been used to treat two
types of syndrome:
– Adenosine deaminase severe combined
– X-chromosome-linked SCID (SCID-X1)
First clinical trial for treatment of an inherited
Ex vivo approach.
ADA cDNA introduced into T lymphocytes by
retroviral-mediated gene transfer.
Gene-corrected cells infused back into patient.
• Gene therapy treatment in combination
with standard ADA injections.
• Restored immune system.
Hematopoietic stem cells treated ex vivo with
retroviral vector carrying the c-receptor cDNA.
Three children developed fatal leukemia.
Activation of the LMO2 proto-oncogene.
LMO2 is a transcription factor required for
hematopoiesis (maturation of blood cells)
Cystic fibrosis gene therapy
• The most common mutation in the cystic
fibrosis transmembrane conductance
regulator (CFTR) gene is a deletion of
three base pairs.
Human clinical trials:
AAV-mediated gene transfer
Although adenovirus- and liposome-mediated
gene therapy were both effective in “curing”
CFTR knockout mice, similar techniques
proved ineffectual in cystic fibrosis patients:
– Administered by a nasal spray.
– Immune response.
– Lack of sustained expression.
Adeno-associated virus (AAV)-mediated
Nasal cells showed CFTR mRNA expression.
cAMP-activated chloride channel function.
What is a potential problem with the AAV
• Twenty years after the discovery of the
cystic fibrosis gene, there is still no cure
for the disease.
HIV-1 gene therapy
• Strategies aim to reduce viral load and
improve patient quality of life.
• Experiments in cell culture are promising,
but much remains to be done before antiHIV-1 gene therapy reaches routine
• Antisense ribozymes have been designed
to specifically target HIV-1RNA
transcripts at different points in the viral
HIV-1 life cycle
Seven major steps in life cycle:
1. Virus-receptor interaction and viral entry.
2. Reverse transcription.
3. Proviral integration.
4. Transcription of viral RNA.
5. Splicing, nuclear export, and translation.
6. Virus particle assembly.
7. Release and viral maturation.
The future of gene therapy
• Recent success story for treatment of X-linked
adrenoleukodystrophy, a severe
• Two boys showed stable expression of the
therapeutic gene and cerebral demyelination
• Two years later, still no sign of progressive brain
• Another recent success: treatment of Leber’s
congenital amaurosis, a form of blindness.
• One eye of patients was injected with a viral
vector carrying a gene coding for an enzyme
needed to make light-sensing pigments.
• Light sensitivity was increased in all 12 children
treated and four children gained enough vision
to take part in normal childhood activities such
as playing catch.