1. Cellular oncogenes

2. Transfection of DNA provides a strategy for detecting nonviral oncogenes
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
(NIH3T3 cells)
transformed by
3-methycholanthrene

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

4. Results of recent transfection assay
Ectopic expression of
telomerase 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

5. Homology between transfected oncogenes and 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

6. ONCOGENES, PROTOONCOGENES, AND THEIR FUNCTIONS
Oncogenes can be classified into five groups based on functional and biochemical properties of their normal counterparts (proto-oncogenes).
growth factors
growth factor receptors
signal transducers
transcription factors
others, including programmed cell death regulators.

7. GROWTH 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 .

8. GROWTH FACTORS
Reciprocal translocation involving chromosomes 17 and 22
Subsequent gene transfer experiments of DPs genomic DNA into NIH 3T3 cells revealed
autocrine activation of the endogenous PDGF receptor by the human rearranged PDGF-b gene
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

9. GROWTH 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.

10. Constitutive activation or over-activation of the erb B
Receptor deletion of the ligand-binding domain
Point mutation in the tyrosine kinase domain
Point mutation in the extracellular domain
Constitutive activation or over-activation of the erb B
deletion of intracellular regulatory domains
Increased expression through gene amplification
abnormal expression in the wrong cell type
Erb B = epidermal growth factor receptor)

11. SIGNAL TRANSDUCERS
Many protooncogenes are members of signal transduction pathways. Consist of two main groups:
Nonreceptor protein kinases
tyrosine kinases (abl, lck, and src)
Serine/threonine kinases (raf-1, mos, and pim-1)
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.

12. TRANSCRIPTION 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.

13. TRANSCRIPTION 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.

14. PROGRAMMED CELL DEATH REGULATION
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

15. MECHANISMS OF ONCOGENE ACTIVATION
Three genetic mechanisms activate oncogenes in human neoplasms:
Mutation
gene amplification
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.

16. Usually involve critical protein regulatory regions
MUTATION
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

17. MUTATION
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.

18. 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

20. Constitutive activation of the signal-transduction
Point mutations in the ras family of proto-oncogenes (K-ras, H-ras, and N-ras)
Carcinogen exposure.
Ras mutations
The majority involve codon 12 of the gene, a smaller number involving other regions such as codons 13
Constitutive activation of the signal-transduction

21. A point mutation is responsible for H-ras oncogene activation
Figure The Biology of Cancer (© Garland Science 2007)

22. GENE 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.

23. 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.

24. The myc oncogene can arise via at least three distinct mechanisms
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.
Figure 4.11a The Biology of Cancer (© Garland Science 2007)

25. erbB2/HER2/neu oncogene can be amplified or overexpressed in human breast carcinoma cells
erbB2/neu oncogene is amplified
erbB2/neu mRNA is overexpressed
increased level of erbB2/neu-encoded protein

26.  Oncogene Amplification in Human Cancers

27. CHROMOSOMAL REARRANGEMENTS
Recurring chromosomal rearrangements are often detected in hematologic as well as in some solid malignancies
mainly of chromosomal translocations
less frequently, chromosomal inversions.
Chromosomal rearrangements can lead to hematologic malignancy via two different mechanisms:
The transcriptional activation of proto-oncogenes
Chromosomal 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.

28. CHROMOSOMAL REARRANGEMENTS
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).

29. Table 4.5 The Biology of Cancer (© Garland Science 2007)

30. Gene transcriptional Activation examples:
The t(8;14)(q24;q32) translocation, is found in about 85% of cases of Burkitt lymphom
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

31. Gene transcriptional Activation 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 a 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.

32. 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.

33. ABL = Abelson murine leukemia virus BCR = breakpoint cluster region
Figure 6-7 .
Gene fusion. The t(9;22)(q34;q11) translocation in chronic myelogenous leukemia (CML) determines the fusion of the c-abl gene with the bcr gene. Such a gene fusion encodes an oncogenic chimeric protein of 210 kDa. Chr = chromosome.
ABL = Abelson murine leukemia virus
BCR = breakpoint cluster region

34. Bcr-Abl fusion protein
Different breakpoints in bcr results in different types of human leukemia
Abl a. b. c.
Bcr
Bcr-Abl fusion protein a b c
acute lymphocytic leukemia
chronic myelogenous
leukemia
chronic neutrophilic leukemia
Figure 4.15b The Biology of Cancer (© Garland Science 2007)