1 molecular biology of cancer cellular oncogenes

1Molecular Biology of Molecular Biology of CancerCancer

Cellular oncogenesCellular oncogenes


(NIH3T3 cells)transformed by

3-methycholanthrene

• Transfection: DNA-mediated transformation technique. This technique, also called gene transfer or transfection assay.

• Verifies the ability of donor DNA from a tumor to transform a recipient strain of cells

Transfection of DNA provides a Transfection of DNA provides a strategy for detecting nonviral strategy for detecting nonviral

oncogenesoncogenes


(NIH3T3 cells)

DNA from tumor cells

The DNA from chemical-transformed tumor cells was able to convert non-tumorigenic NIH3T3 fibroblasts into tumorigenic cells.


Results of recent transfection assay Results of recent transfection assay

Ectopic expression oftelomerase catalytic subunit (hTERT) + SV40 large T product + mutant H-ras oncogenic direct tumorigenic conversion of normal human epithelial and fibroblast cells.

Many of the oncogenes identified by gene-transfer studies are identical or closely related to those oncogenes transduced by retroviruses.Members of the ras family have been

repeatedly isolated from various human tumors by gene transfer.

Recently, neu, met, and trk


Homology between transfected Homology between transfected oncogenesoncogenes

and retroviral oncogenesand retroviral oncogenes

Figure 4.5 The Biology of Cancer (© Garland Science 2007)

NIH3T3 cell lines transfected with DNA extracted from a human

bladder carcinoma cell line

Untransfected NIH3T3

Probe used: H-ras oncogene present in Harvey rat sarcoma virus


ONCOGENES, PROTOONCOGENES, ONCOGENES, PROTOONCOGENES, AND THEIR FUNCTIONSAND THEIR FUNCTIONS

Oncogenes can be classified into five groups based on functional and biochemical properties of their normal counterparts (proto-oncogenes).1.growth factors2.growth factor receptors3.signal transducers4.transcription factors5.others, including programmed cell death

regulators.


GROWTH FACTORSGROWTH FACTORS

Example: sis oncogene of simian sarcoma virus, a retrovirus first isolated from a monkey fibrosarcoma.Sequence analysis: showed that sis encodes the beta

chain of PDGF.inappropriately expressed growth factors may

function as oncogenes.Constitutive expression of the sis gene product PDGF-ß

neoplastic transformation of fibroblasts containing PDGF receptor No transformation of cells lacking the PDGF receptor.

Thus, transformation by sis requires interaction of the sis gene product with the PDGF receptor by autocrine stimulation .


Reciprocal translocation involving chromosomes 17 and 22

fusion between the collagen type Ia1 (COL1A1) gene and PDGF-ß gene

deletion of PDGF-ß exon 1

a constitutive release of PDGF-ß growth factor

Dermatofibrosarcoma protuberans (DP) is an infiltrative skin tumor

Subsequent gene transfer experiments of DPs genomic DNA into NIH 3T3 cells revealedautocrine

activation of the endogenous PDGF receptor by the human rearranged PDGF- gene

GROWTH GROWTH FACTORSFACTORS


GROWTH FACTOR RECEPTORSGROWTH FACTOR RECEPTORS

Some viral oncogenes are altered versions of normal growth factor (receptor tyrosine kinases).

Growth factor receptors constitute an important class of protooncogenes involved in the regulation of normal cell growth.

Examples include: erb B, erb B-2, fms, kit, met, ret, ros, and trk.

Mutation or abnormal expression of growth factor receptors can convert them into oncogenes.


Constitutive activation or

over-activation of the erb B

Erb B = epidermal growth factor receptor)

Receptor deletion of the ligand-binding domain

Point mutation in the tyrosine kinase domain

Point mutatio

n in th

e

extrace

llular d

omain

deletion of intracellular

regulatory domains

Increased expression through gene amplification

abnormal expression in the wrong cell type


SIGNAL TRANSDUCERS SIGNAL TRANSDUCERS

Many protooncogenes are members of signal transduction pathways. Consist of two main groups:1. Nonreceptor protein kinases

tyrosine kinases (abl, lck, and src) Serine/threonine kinases (raf-1, mos, and pim-1)

2. Guanosine triphosphate (GTP)-binding proteins Monomeric GTP-binding proteins: ras family of protooncogenes (H-

ras, K-ras, and N-ras) Heterotrimeric GTP-binding proteins (G proteins) implicated as

protooncogenes: currently include gsp and gip.

Signal transducers are often converted to oncogenes by mutations that lead to their unregulated activity, which in turn leads to uncontrolled cellular proliferation.


TRANSCRIPTION FACTORSTRANSCRIPTION FACTORS

Many protooncogenes are transcription factors that were discovered through their retroviral homologs

Examples include erb A, ets, fos, jun, myb, and c-myc.Fos + jun AP-1 transcription factor positively

regulates a number of cell division activating genes

Erb A is the receptor for the T3 thyroid hormone, triiodothyronine.


TRANSCRIPTION FACTORSTRANSCRIPTION FACTORS

Protooncogenes that function as transcription factors are often activated by chromosomal translocations in hematologic and solid neoplasms.

Example: c-myc gene:helps to control the expression of genes leading to

cell proliferation.

c-myc gene is frequently activated by chromosomal translocations in human leukemia and lymphoma.


PROGRAMMED CELL DEATH PROGRAMMED CELL DEATH REGULATIONREGULATION

The only protooncogene thus far shown to regulate programmed cell death is bcl-2.Bcl-2 was discovered by the study of

chromosomal translocations in human lymphoma.

bcl-2 activation inhibits programmed cell death in lymphoid cell populations (survival).

bcl-2 is not the only apoptosis gene involved in neoplasia, additional protooncogenes await identification


MECHANISMS OF ONCOGENE MECHANISMS OF ONCOGENE ACTIVATIONACTIVATION

Three genetic mechanisms activate oncogenes in human neoplasms:1. Mutation2. gene amplification3. chromosome rearrangements

These mechanisms result in either an alteration of protooncogene structure or an increase in protooncogene expression

Because neoplasia is a multistep process more than one of these mechanisms often

contribute to the genesis of human tumors by altering a number of cancer-associated genes.


Mutations activate proto-oncogenes through structural alterations in their encoded proteins

Usually involve critical protein regulatory regions

Often lead to the uncontrolled, continuous activity of the mutated protein

MUTATIONMUTATION


MUTATIONMUTATION

Retroviral oncogenes, often have deletions that contribute to their activation.deletions in the amino- terminal ligand-binding

domains of the erb B, kit, ros, met, and trk oncogenes.

In human tumors, most characterized oncogene mutations are base substitutions (point mutations) that change a single amino acid within the protein.


Point mutations in the ras family of proto-oncogenes (K-ras, H-ras, and N-

ras)About 15% to 20% of human tumors may

contain a ras mutation.Mutations in K-ras predominate in carcinomas.

about 30% of lung adenocarcinomas, 50% of colon carcinomas, and 90% of carcinomas of the pancreas.

N-ras mutations are preferentially found in hematologic malignancies up to a 25% incidence in acute myeloid leukemias and

myelodysplastic syndromes.

The majority of thyroid carcinomas have been found to have ras mutations distributed among K-ras, H-ras, and N-ras


Point mutations in the ras family of proto-oncogenes (K-ras, H-ras, and N-

ras)Carcinogen exposure.

The majority involve codon 12 of the gene, a smaller number involving other regions such as

codons 13

Constitutive activation of the signal-transduction

Ras mutations


Figure 4.10 The Biology of Cancer (© Garland Science 2007)

A point mutation is responsible for A point mutation is responsible for H-H-rasras oncogene activation oncogene activation


GENE AMPLIFICATIONGENE AMPLIFICATION

The process of gene amplification occurs through redundant replication of genomic DNA, often giving rise to karyotypic abnormalities:double-minute chromosomes (DMs)

are characteristic minichromosome structures without centromeres

homogeneous staining regions (HSRs) HSRs are segments of chromosomes that lack the normal

alternating pattern of light- and dark-staining bands

up to several hundred copies of a gene.Amplification leads to the increased expression of

genes, which in turn can confer a selective advantage for cell growth.


Studies demonstrated that three protooncogene families (myc, erb B, and ras) are amplified in a significant number of human tumors:

Examples: c-myc amplification: about 20% to 30% of breast and ovarian

cancers; and in some types of squamous cell carcinomas erb B amplification: found in up to 50% of glioblastomas and

in 10% to 20% of squamous carcinomas of the head and neck. erbB-2 (HER-2/neu) gene amplification: Approximately in

15% to 30% of breast and ovarian cancers. Members of the ras gene family, including K-ras and N-ras,

are sporadically amplified in various carcinomas.


Figure 4.11a The Biology of Cancer (© Garland Science 2007)

homogeneous staining regions (HSR)

The N-myc gene amplification is found in 30% of human childhood neuroblastoma.

Astrocytoma, retinoblastoma and small-cell lung carcinomas (neuroendocrinal traits) also often exhibit amplified N-myc genes.

* N-myc is a close relative of c-myc.

The The mycmyc oncogene can arise via oncogene can arise via at least three distinct at least three distinct

mechanismsmechanisms


erbB2/neu oncogene is amplified

erbB2/neu mRNA is overexpressed

increased level of erbB2/neu-encoded protein

erbB2/HER2/neu oncogene can be amplified or overexpressed in human breast carcinoma cells


Oncogene Amplification in Human Cancers Oncogene Amplification in Human Cancers


CHROMOSOMAL REARRANGEMENTS CHROMOSOMAL REARRANGEMENTS

Recurring chromosomal rearrangements are often detected in hematologic as well as in some solid malignancies mainly of chromosomal translocationsless frequently, chromosomal inversions.

Chromosomal rearrangements can lead to hematologic malignancy via two different mechanisms:

1.The transcriptional activation of proto-oncogenesChromosomal rearrangements move a proto-oncogene

close to an immunoglobulin or T-cell receptor gene Transcription of the protooncogene then falls under control

of regulatory elements from the immunoglobulin or T-cell receptor locus.


CHROMOSOMAL REARRANGEMENTS CHROMOSOMAL REARRANGEMENTS

2. The creation of fusion genes. Fusion genes can be created by

chromosomal rearrangements when the chromosomal breakpoints fall within the loci of two different genes.

Fusion genes encode chimeric proteins with transforming activity.

In some cases, the same protooncogene is involved in several different translocations (c-myc, ews, and ret).


• The expression of c-myc gene is placed under control of the trancription-controlling enhancer sequences of an immunoglobulin heavy chain (IgH) gene.

• c-myc encodes a nuclear protein involved in the regulation of cell proliferation

1.1. Gene transcriptional Activation Gene transcriptional Activation examples: examples:

• The t(8;14)(q24;q32) translocation, is found in about 85% of cases of Burkitt lymphom


1.1. Gene transcriptional Activation Gene transcriptional Activation examples: examples:

In addition to c-myc, several proto-oncogenes that encode nuclear proteins are activated by various chromosomal translocations in T-ALL involving the T-cell receptor or ß locus.

These include HOX11, TAL1, TAL2, and RBTN1/Tgt1.The proteins encoded by these genes are thought

to function as transcription factors through DNA-binding and protein-protein interactions.


2.2. Gene Fusion Examples: Gene Fusion Examples:

The first example of gene fusion was discovered through the cloning of the breakpoint of the Philadelphia chromosome in chronic myelogenous leukemia (CML).

The t(9;22)(q34;q11) translocation in CML fuses the c-abl gene, normally located at 9q34, with the bcr gene at 22q11 The bcr/abl fusion, created on the der(22) chromosome,

encodes a chimeric protein of 210 kDa increased tyrosine kinase activity and abnormal cellular localization.

The t(9;22) translocation is also found in up to 20% of cases of acute lymphoblastic leukemia (ALL). In these cases, the breakpoint in the bcr gene differs

somewhat from that found in CML, resulting in a 185 kDa bcr/abl fusion protein.


ABL = Abelson murine leukemia virus

BCR = breakpoint cluster region


Figure 4.15b The Biology of Cancer (© Garland Science 2007)

acute lymphocytic leukemia

chronic myelogenousleukemia

chronic neutrophilic leukemia

Different breakpoints in Different breakpoints in bcr bcr results results in different types of human leukemiain different types of human leukemia

Abl

a.

b.

c.

Bcr

Bcr-Abl fusion protein

a b c

1 molecular biology of cancer cellular oncogenes

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pdgf gene deletion of

tumor cells

transformation of cells

transfected oncogenes

tumorigenic cells

nonviral oncogenes

functions oncogenes

endogenous pdgf receptor