Chapter 17:

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

17.1 Introduction

• Discoveries in molecular biology have led to many advances in medicine.

• Increased understanding of the nature of human disease.

• Development of treatment strategies.

17.2 Molecular biology of cancer

“If thou examinest a man having tumors on his breast… There is no treatment.”

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 the USA?

• One half of all men.

• One third of all women.

Cancer is a multistep disease

• Not just one disease but a group of genetically diverse disorders.

• Each tumor can have its own “genetic signature.”

• 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 acquired.

Three major changes that occur when a cell becomes cancerous:

• Immortalization

• Transformation

• Metastasis

Genetic changes associated with tumorigenesis:

• Gain of function– Inappropriate activation of oncogenes

• Loss of function– Inactivation of tumor suppressor genes

Activation of proto-oncogenes and oncogenes

• Oncogenes are genes whose products have the ability to cause malignant transformation of eukaryotic cells.

• 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 were isolated.

• Designated by “v-” to indicate their original identification in retroviruses.– e.g. v-src, v-myc

• “c-” signifies the cellular proto-oncogene counterpart.

Inappropriate activation of proto-oncogenes 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 and metastasis.

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 cell growth.

• Cancer arises when they are not expressed.

Two well-characterized tumor suppressor gene products:

• Retinoblastoma protein (pRB)

• p53 protein

Knudson’s two-hit hypothesis and retinoblastoma

• 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 master switch

• First tumor suppressor protein gene to be cloned.

• Deletion of the gene is linked to retinoblastoma, a childhood hereditary cancer syndrome resulting in tumors of the retina.

• The cell cycle is driven by the coordinated activation of cyclin-dependent 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 transcription factors.

• 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 degradation.

• 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 cycle:

• 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 p53.

The discovery of p53

• Researchers first concluded that p53 was an oncogene

• A p53 cDNA clone derived from “normal” liver cells turned out to be a mutant form of p53 with transforming activity.

• Wild-type p53 was shown not to have transforming activity.

Inappropriate expression of microRNAs in cancer

• 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.

Example:

• OncomiRs – oncogenic microRNAs.

• Overexpression of the mir-17-19b miRNA gene cluster accelerates c-myc-induced B cell lymphoma.

Chromosomal rearrangements and cancer

• 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 protein.

• 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 infection:

– 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 verruciformis.

• Viral protein E6 inactivates p53.

• E6 also activates transcription of the gene encoding the reverse transcriptase subunit of telomerase.

• 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 an oncogene

• Many retroviruses lose part of their genome during rearrangement

• 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 carcinogenesis

• Chemical carcinogens or their metabolic products can either directly or indirectly affect gene expression:

– Genotoxic effects

– Nongenotoxic effects

Genotoxic effects

• Benzo(a)pyrene in cigarette smoke induces:

– The formation of DNA adducts that interfere with replication and transcription.

– Chromosomal rearrangements.

Nongenotoxic effects

• 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 complete regression.

RNAi therapies

• Goal: knockdown of disease-related gene expression in humans.

• First clinical trial: treatment of macular degeneration.

• Animal model: RNAi can lower cholesterol levels in mice.

Vectors for somatic cell gene therapy

• Two main strategies for somatic cell gene therapy:

– 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 expression.

– Immune response.– Activation of an oncogene.

• The majority of gene therapy trials use viral vectors.

• Use of liposomes and naked DNA has been explored.

Three main viral vectors for somatic cell gene therapy:

• 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 viral RNA.

• 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 therapeutic option.

• Treatment of Jesse Gelsinger for partial ornithine transcarbamylase (OTC) deficiency.

• Injection of recombinant adenovirus into portal vein.

• 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 immunodeficiency syndromes

• Gene therapy has been used to treat two types of syndrome:

– Adenosine deaminase severe combined immunodeficiency (ADA-SCID)

– X-chromosome-linked SCID (SCID-X1)

ADA-SCID

• First clinical trial for treatment of an inherited disorder.

• 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.

SCID-X1

• 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 gene transfer:

• Aerosol administration.

• Nasal cells showed CFTR mRNA expression.

• cAMP-activated chloride channel function.

• What is a potential problem with the AAV vector?

• 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 anti-HIV-1 gene therapy reaches routine clinical practice.

Anti-HIV-1 ribozymes

• Antisense ribozymes have been designed to specifically target HIV-1RNA transcripts at different points in the viral life cycle.

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 neurodegenerative disorder.

• Two boys showed stable expression of the therapeutic gene and cerebral demyelination was arrested.

• Two years later, still no sign of progressive brain damage.

• 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.