Molecular Biology for Urologists

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Practical Urology: EssEntial PrinciPlEs and PracticE

Table 12.1. important definitions and concepts in molecular biology

Term

Section

Definition

tumor suppressor gene

a

a gene encoding a protein which when lost tends to result in the formation of a

neoplasm

genome

a

all the genes found within a given organism

somatic cell

a

the cells within a multicellular organism that do not produce gametes (oocyte

or sperm).these represent the vast majority of cells in an organism

germ line mutations

a

a permanent, heritable change in the dna sequence within the genome of a

gamete (oocyte or sperm). as a result, it is present in every cell of any

organism which develops from that cell

chromosome

a

the structure within the cell nucleus that stores the dna in linear strands

associated with a variety of proteins such as histones

genetic changes

B

direct mutations or alterations in the dna sequence that are passed on from

parent to progeny cells

Epigenetic changes

B

Heritable changes in the expression of genes which do not involve a direct dna

mutation. an example is dna methylation

codon

B

a sequence of three adjacent nucleotides that codes for a particular amino acid.

the sequence of these successive codons specifies the amino acid sequence

of the resulting protein

Frame shift mutation

B

a mutation (or change in dna) in which nucleotides are inserted or deleted

such that the downstream sequence of codons is scrambled. since dna is

read three nucleotides at a time (see codon above) a shift in the nucleotide

sequence puts the entire remaining sequence out of frame, resulting in a

scrambled and nonfunctional protein

nonsense mutation

B

a mutation (or change in dna) in which an early or premature stop codon is

created, resulting in a truncated protein that is typically nonfunctional.

Missense mutation

B

a mutation (or change in dna) in which single nucleotide within a codon is

changed resulting in a single amino acid change in a protein.this may or

may not result in altered function

dna methylation

B

Modification of dna in which a methyl group (cH3) is added to a cytosine that

immediately precedes a quanine (cpg island).this is carried out by enzymes

called dna methyl transferases (dnMt)

Promoter

B

the site in the dna associated with a particular gene where the proteins

responsible for transcription bind to initiate mrna expression. as a result,

this site is where the expression of that particular gene is controlled

response element

c

the dna nucleotide sequence in the promoter of a gene that is recognized by

a regulatory protein that controls transcription, such as the HiFa/b

heterodimer. Binding of the regulatory protein to its response element within

a particular gene will either upor downregulate expression of that gene

Messenger rna (mrna)

c

a single stranded rna molecule, coded for by the dna of a gene, which specifies

the subsequent amino acid sequence of a protein during protein synthesis

transcription

c

the process by which the genetic code in dna is transcribed into a complemen-

tary, single stranded mrna molecule for subsequent protein synthesis

translation

c

the process whereby the genetic code stored in the transcribed mrna molecule

is now translated into a sequence of amino acids to form a protein.this is

accomplished by the cooperative work of the ribosomes and trna that is

associated with specific amino acids

Table 12.1. (continued)

Term

Section

Definition

oncogene

c

a gene that is mutated or overexpressed in a cell, allowing it to become

neoplastic or malignant

Polymerase chain

c

a technique to significantly amplify a specific stretch of dna using a set of dna

reaction (Pcr)

primers designed to specify an area of interest

rt-Pcr

c

a variation of Pcr in which mrna is reverse transcribed into dna and then Pcr

is done on a gene of interest.this allows the scientist to measure gene

expression at the mrna level

genomics

c

a large-scale, rapid screen of gene expression across the entirety of the cell’s

dna, that is, its genome

Proteomics

c

a large-scale, rapid screen of the level of all the proteins within a cell at a given

time

angiogenesis

d

the process by which new blood vessels are formed

ligand

d

a molecule, typically a soluble one such as a hormone, which binds to another

molecule to initiate a subsequent downstream effect

receptor

d

a molecule within or on the surface of a cell that when it binds its specific ligand

(see above) it results in specific,downstream effects important to cellular function.

Kinase

d

a frequently used abbreviation for a phosphokinase.these are enzymes which

transfer a phosphate group from atP to another molecule

cytoplasm

d

the portion of a eukaryotic cell that lies between the outer cellular surface

membrane and the inner nucleus. it houses much of the cell’s physiologic

machinery and organelles, such as the mitochondria, ribosomes, golgi

apparatus, and endoplasmic reticulum

nucleus

d

the large organelle that houses the genomic dna in eukaryotic cells within

chromosomes

Phosphorylation

d

the process whereby phosphate bonds are created between two substrates. in

cells this is accomplished usually by kinases

tyrosine kinase

d

an enzyme that transfers a phosphate group from atP to a tyrosine residue,

either within its own structure or another molecule

targeted therapy

E

a form of therapy that is designed to specifically target molecules important to

the growth of cancer cells, rather than just target rapidly dividing cells such as

cytotoxic therapy (i.e., traditional chemotherapy). Examples include

antibodies to specific molecules and small molecule kinase inhibitors

Monoclonal antibody

E

an antibody that can be produced in large quantities in a pure form from a

single cell clone. Each antibody molecule is identical with respect to its

structure and specificity for a particular target (termed an antigen)

tyrosine kinase inhibitor

F

a small molecule that interferes with the tyrosine kinase activity of enzymes in

cells.this is a prototypical example of a targeted therapy

cell cycle

g

the regulated sequence by which cells undergo the process of growth and cell

division/replication

apoptosis

also termed programmed cell death, it is the active, regulated process by which

cells die in response to external or internal stimuli

Heterodimer

the association between two different proteins to form a complex. an example is the

association between HiFaand HiFbin response to hypoxia or loss of VHl protein

Homodimer

the association between two identical proteins to form a complex. an example

is the association between two androgen receptor molecules within a cell

164

Practical Urology: EssEntial PrinciPlEs and PracticE

order for a cancer to develop. In a sporadic, non-

What followed was based on the recognition

inherited form of cancer, this means that for any

that these sporadic clear cell RCCs were also

given cell in the body, a mutation is needed in

seen in von Hippel-Lindau disease and the

both copies of the same gene. This is because

abnormality on chromosome 3p fit the profile of

any mutation or change in the genome would be

a putative tumor suppressor gene. In a series of

in a somatic cell, and so, only that particular cell

elegant and groundbreaking studies of multiple

and its progeny would have that change, whereas

different families with von Hippel-Lindau dis-

the remaining cells in the body would not. It

ease, the localization of the VHL gene was

would be a rare event that each copy of a par-

mapped to a relatively small region on chromo-

ticular gene would acquire a mutation in the

some 3p,18–20, which was then followed by the

same cell; so in sporadic, nonfamilial cancers,

identification of the VHL gene in a seminal arti-

the tumors generally occur later in life and are

cle by Latif et al. in 1993.21 Since that time, it has

usually unifocal. On the other hand, in inherited

been demonstrated that the majority of sporadic

familial tumor syndromes, there is a germ line

clear cell RCC tumors harbor aberrations of

mutation in one tumor suppressor gene. Thus,

VHL, strongly suggesting that the same gene

every cell in the body has inherited a nonfunc-

was responsible for both the inherited and non-

tional copy of the gene (the first hit). As a

inherited forms of the disease.1,2,22–26 Indeed,

consequence, now it only takes one additional

even in cases where mutations of VHL were not

mutation of the same gene (the second hit) in

identified, most often other aberrations were

one cell in order for a cancer to develop. Since

noted such as abnormal hypermethylation of

every cell already has the first hit, having a sec-

the promoter region of VHL, leading to low or

ond hit would be more common. Patients suffer-

absent protein levels (see below).27

ing from such inherited tumor syndromes

Since the discovery of VHL, its role in the nor-

would, therefore, develop cancers in the affected

mal function of the cell and how it can act as a

organs at a younger age and tend to have multi-

tumor suppressor gene has been carefully stud-

focal disease. The basic tenets of this hypothesis

ied.The principles illustrated here are applicable

were developed in the 1980s for familial tumor

to a host of other disease processes and serve as

syndromes such as retinoblastoma and the Rb

an excellent example of how the discoveries at

gene and neurofibromatosis and the NF-1 and

the bench have been translated to the bedside in

NF-2 genes.3–7 In urology, the classic example of

the form of new targeted agents for advanced

this is von Hippel-Lindau disease, the VHL gene,

RCC. It is instructive, therefore, to describe this

and the development of clear cell RCC.

in some detail, though the interested reader is

The discovery of the VHL gene and its rele-

encouraged to read any one of the many in-

vance to clear cell RCC grew at first from the

depth reviews on this topic.

observation that the RCC in von Hippel-Lindau

disease was identical to its sporadic, nonfamilial

Inherited Changes in Cancer Cells

counterpart in every respect except it occurred

earlier in life and tended to be multifocal. It

therefore fit the profile of a familial tumor syn-

It is worth pausing for a moment to review vari-

drome characterized by the loss/mutation of a

ous ways in which a gene, such as VHL, can

tumor suppressor gene. Careful studies of three

become aberrantly regulated. A critical element

different families (kindreds) with a familial ten-

for all of these mechanisms is that they can be

dency toward the development of clear cell RCC

inherited or passed on to any progeny cells.

had consistently showed aberrations of the short

Thus, once a “parent” or originating cancer cell

arm of chromosome 3 (termed 3p).8–10 This

acquires one of these changes, those changes are

was followed by a series of studies of clear cell

passed on to all its progeny resulting in the

RCC tumors and cell lines that also showed

growth of a tumor. The two fundamental forms

abnormalities of chromosome 3p as a unifying

of such changes are genetic changes, such as

theme.11–15 The changes noted in chromosome 3

direct mutations of DNA,and epigenetic changes,

were not present in normal tissues in these spo-

inherited alterations in how genes are expressed

radic clear cell RCC tumors and were not pres-

(such as DNA methylation). Both of these mech-

ent in other histologic RCC variants (such as

anisms can be demonstrated in the VHL gene

papillary RCC).16,17

and clear cell RCC.

Genetic changes (mutations of the DNA in the

them (de-acetylated via enzymes called histone

genome) are inherited from one cell and passed

de-acetylases or HDACs) or methyl groups

on to all its progeny. If that cell is a gamete, such

added (methylated via enzymes called histone

as an egg or sperm, then this mutation will be

methyltransferases, HMTs). In both cases, these

found in every cell in the new organism. These

can cause gene expression to be silenced.Perhaps

are the types of mutations found in familial

the most intensely studies form of epigenetic

tumor syndromes such as von Hippel-Lindau

change as it relates to cancer is the silencing of

Disease. If a mutation occurs in a non-gamete

gene expression via DNA methylation. This

cell (i.e., any cell after conception), then this

involves the covalent bonding of a methyl group

somatic mutation is passed on only to that cell’s

to a cytosine in areas where there is a cytosine

progeny. Furthermore, such a somatic mutation

immediately followed by a guanine, a sequence

will not be passed on from generation to genera-

known as a CpG island. This is carried out by a

tion. These are the mutations found in sporadic

class of enzymes called DNA methyltransferases

cancers. The types of mutations that can occur in

(DNMTs). When these CpG islands are methy-

the DNA include insertions or deletions of small

lated in the promoter of a gene, the gene’s expres-

or large portions of the DNA. Since every three

sion is often shut down. This mechanism is

DNA nucleotides represents one codon (coding

relevant to clear cell renal cell carcinoma, in that

element for one amino acid), if the insertion or

a significant fraction of sporadic RCCs have

deletion occurs in a multiple of three, the overall

been shown to lose VHL protein through DNA

protein function may or may not be disrupted

methylation and gene silencing, an epigenetic

depending on what amino acid(s) is inserted/

change, rather than by a mutation.27 There is

deleted. If the number of nucleotides inserted/

ongoing and intensive interest in the therapeutic

deleted is not a multiple of three, then the entire

potential of HDAC and DNMT inhibitors across

sequence of nucleotide codons and resulting

a variety of tumor types, though their direct

amino acids will be altered resulting in a frame

clinical utility in RCC remains to proven.

shift mutation. This will, in effect, result in a

scrambled series of amino acids and a nonfunc-

The Tumor Suppressor VHL,

tional protein. Roughly, half the VHL mutations

found in sporadic clear RCC are of this type.22

Oncogene HIF, and Gene

This will result in a completely nonfunctional

Regulation

protein. In some cases, the nucleotide may be

mutated in a way that places a premature stop

codon, so a portion of the protein is truncated.

In a normal cell, the VHL protein acts predomi-

These are referred to as nonsense mutations. In

nantly to regulate the cell’s response to the local

other cases, one nucleotide is mutated/changed

availability of oxygen.33–38 When local oxygen

such that only one amino acid is altered. This is

levels are normal, there is another regulatory

termed a missense mutation and represents the

protein, termed hypoxia inducible factor alpha

majority of the remaining VHL mutations in

(HIFa), which is hydroxylated permitting it to

sporadic, clear cell RCCs.22 A more in-depth

bind to the VHL protein (see Fig. 12.1).39,40 The

analysis of the specific VHL mutations and their

VHL protein is actually part of an enzyme com-

potential relationship to disease biology has

plex (called an E3 ligase) that joins a series of

been reviewed previously.28,29

molecules to HIFa called ubiquitin, which in

The expression of genes can also be altered

turn marks HIFa for degradation.41–46 In the

via mechanisms that do not involve a mutation

normal circumstance, then, HIFa levels are low

in DNA. If these alterations are heritable, then

in the cell. During hypoxia when oxygen levels

these are termed epigenetic changes.30–32 The

in the cell are low, HIFa is not hydroxylated, so

ability to silence genes is critical to the normal

it does not bind to VHL protein, and conse-

development of an organism. There are a num-

quently is not degraded. The levels of HIFa in

ber of mechanisms by which this can be accom-

the cell rise, allowing it to bind with a similar

plished. This includes modifications of the

molecule, HIF-b, that exists at high levels in the

proteins that are involved in packaging DNA

cell at all times (see Fig. 12.2). This complex can

within the nucleus of cells called histones. These

then bind to specific regions in the cell’s nuclear

histones can have acetyl groups removed from

DNA called hypoxia response elements (HRE).