1Molecular Biology of Molecular Biology of CancerCancer
Cellular oncogenesCellular oncogenes
2Molecular Biology of Molecular Biology of CancerCancer
(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
3Molecular Biology of Molecular Biology of CancerCancer
(NIH3T3 cells)
DNA from tumor cells
The DNA from chemical-transformed tumor cells was able to convert non-tumorigenic NIH3T3 fibroblasts into tumorigenic cells.
4Molecular Biology of Molecular Biology of CancerCancer
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
5Molecular Biology of Molecular Biology of CancerCancer
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
6Molecular Biology of Molecular Biology of CancerCancer
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.
7Molecular Biology of Molecular Biology of CancerCancer
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 .
8Molecular Biology of Molecular Biology of CancerCancer
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
9Molecular Biology of Molecular Biology of CancerCancer
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.
10Molecular Biology of Molecular Biology of CancerCancer
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
11Molecular Biology of Molecular Biology of CancerCancer
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.
12Molecular Biology of Molecular Biology of CancerCancer
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.
13Molecular Biology of Molecular Biology of CancerCancer
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.
14Molecular Biology of Molecular Biology of CancerCancer
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
15Molecular Biology of Molecular Biology of CancerCancer
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.
16Molecular Biology of Molecular Biology of CancerCancer
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
17Molecular Biology of Molecular Biology of CancerCancer
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.
18Molecular Biology of Molecular Biology of CancerCancer
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
19Molecular Biology of Molecular Biology of CancerCancer
20Molecular Biology of Molecular Biology of CancerCancer
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
21Molecular Biology of Molecular Biology of CancerCancer
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
22Molecular Biology of Molecular Biology of CancerCancer
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.
23Molecular Biology of Molecular Biology of CancerCancer
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.
24Molecular Biology of Molecular Biology of CancerCancer
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
25Molecular Biology of Molecular Biology of CancerCancer
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
26Molecular Biology of Molecular Biology of CancerCancer
Oncogene Amplification in Human Cancers Oncogene Amplification in Human Cancers
27Molecular Biology of Molecular Biology of CancerCancer
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.
28Molecular Biology of Molecular Biology of CancerCancer
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).
29Molecular Biology of Molecular Biology of CancerCancer
Table 4.5 The Biology of Cancer (© Garland Science 2007)
30Molecular Biology of Molecular Biology of CancerCancer
• 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
31Molecular Biology of Molecular Biology of CancerCancer
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.
32Molecular Biology of Molecular Biology of CancerCancer
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.
33Molecular Biology of Molecular Biology of CancerCancer
ABL = Abelson murine leukemia virus
BCR = breakpoint cluster region
34Molecular Biology of Molecular Biology of CancerCancer
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