B. Steps of signal transduction

A. Definitions and description

1. Retroviral oncogenes (v-onc) were first discovered in rapidly transforming retroviruses extracted from animal tumors. Approximately 20 oncogenes of this type have been described.

2. Cellular oncogenes were discovered by extracting DNA from cancer cells and inserting it into normal cells, which then become malignant (DNA transfection). More than 20 oncogenes of this type have been described.

3. Inhibitory genes produce proteins that inhibit cell proliferation. Abnormalities in these genes can lead to abnormal proliferation of cells. About 12 such genes have been described.

4. Proto-oncogenes (c-onc or normal cell genes) control proliferation and differentiation in normal cells and appear to be the source of all oncogenes. RNA from transforming retroviruses is homologous with various proto-oncogenes, which is evidence for this conclusion.

a. Proto-oncogenes are highly conserved through evolution. Many of the same oncogenes are found in life forms as diverse as humans and yeast. They appear to be essential to life.

b. Each proto-oncogene makes protein products that are differentially expressed during the cell cycle or at specific stages of development of a particular tissue.

5. Differences between c-onc and proviral v-onc

a. Cellular oncogenes have both introns and exons, and viral oncogenes have only exons.

b. Proviral DNA possesses LTRs, which are powerful promoter genes not found in c-onc. When LTRs are inserted near a c-onc, they can deregulate it and transform the cell into a malignant phenotype.

A model for understanding oncogene biology

Step 1: Growth factors of both proto-oncogene and non-proto-oncogene origin combine with specific growth factor receptors (GFRs) on target cells, activating tyrosine kinase activity on the GFR (see step 2). The growth factor-GFR complex itself is taken up by the cell and deactivated. Some growth factors and their associated proto-oncogenes are fibroblast growth factors (Int-2 and others) and platelet-derived growth factors (sis).

Example: sis oncogene

a. Normal function. The sis oncogene codes for one of the chains of platelet-derived growth factor (PDGF). PDGF is manufactured in megakaryocytes and packaged in platelets. When platelets become activated, they release PDGF, which stimulates its surface receptor to produce tyrosine kinase activity and stimulates the proliferation of fibroblasts.

b. Abnormalities in cancer cells involve unregulated production due to ectopic location of gene, possible amplification, and truncated protein.

c. Associated human cancers include some squamous cancers, glioblastomas, acute myelogenous leukemia (AML), and osteosarcomas. The sis oncogene is possibly activated by HTLV-1.

d. Effect of abnormalities on prognosis. None are known.

Step 2. Growth factor receptors (GFRs) and some hormone receptors are proto-oncogene proteins that traverse the cell membrane. The cell surface part of the protein has sites for specific extracellular growth factors. When GFRs are activated by their growth factors, their cytoplasmic sites become active tyrosine kinases. Some GFRs and their proto-oncogenes are colony-stimulating factor receptor-1 (fms) and epidermal growth factor receptor (erb -B).

The activated tyrosine kinases transduce the extracellular signal to cytoplasmic proteins and to the nucleus by a variety of mechanisms. For example, they activate cytoplasmic proto-oncogene proteins. They also increase the levels of diacylglycerol, which then activate protein kinase C (PKC); PKC activates a number of proteins that stimulate DNA synthesis. They also phosphorylate guanosine 5’-diphosphate (GDP) to make the triphosphate (GTP), which activates p21 (a c-ras protein); the GTP-p21 complex stimulates DNA transcription in the nucleus.

Example: erb -B oncogene (Others: fms, ros, sea)

a. Normal function. The proto-oncogene counterpart of erb -B produces cell surface receptors for EGF and has tyrosine kinase activity when activated by EGF.

b. Abnormalities in cancer cells. The erb -B gene product produces a truncated product with unregulated protein kinase activity. It delivers a constant proliferative signal to the cell.

c. Associated human cancers are squamous cancers and glioblastomas.

d. Effect of abnormalities on prognosis. Survival is poor with increased expression of erb -B for patients with breast cancer, upper respiratory tumors, and uterine cervix cancer.

Step 3. Cytoplasmic kinases that modulate the activity of key cellular enzymes are activated by membrane receptor kinases. These proteins typically have serine or threonine kinase phosphorylating activity and some have GTPase activity. These kinases have an extensive variety of effects in activating nuclear proteins and in direct activation of DNA promoter regions. For example, the cell division cycle kinase (cdc) is found in all eukaryotic cells and is essential to the transition of cells from the G₂ phase to the M phase. Its activity is regulated by cyclin proteins and phosphorylation and it appears to act by destabilizing the cytoskeleton in preparation for mitosis.

Example: ras oncogene (Others: raf, mos, src, yes, fps, abl, crk, cdc)

a. Normal function. Proto-oncogene precursors of c-ras proteins transduce signals from the cell surface to the nucleus. The proteins combine with GDP and GTP. The protein-GTP complex (e.g., p21-GTP) is the chemically active form, which then activates a variety of nuclear promoter proteins. The protein has GTP-ase activity that is enhanced by a cytoplasmic protein called GAP. The GTP-ase converts its own bound GTP to GDP, inactivating the complex; this phenomenon is thought to be a self-regulatory activity of the protein. The ras protein also increases levels of diacetylglycerol, which activates PKC, an important initiator of DNA transcription (see step 4).

b. Abnormality in cancer cells. The ras oncogenes are a group of five related oncogenes with most of the activities of their normal proto-oncogene counterparts. Several defects, including defective GTPase, have been found. This abnormality leads to a constant presence of the GTP form of the molecule, which continuously activates nuclear proteins and DNA transcription.

c. Associated human cancers. These include AML, some melanomas, neuroblastomas, breast cancers, bladder cancers, and lung cancers.

d. Effect of abnormalities on prognosis. The prognosis is poor for AML.

Step 4. Proteins directly controlling gene expression («transcription factors») are involved in the final step of signal transduction. Some of these factors are short-lived and never leave the nucleus. For example, c-jun and c-fos proteins are among the first proteins transcribed at the G₀ -S interface and appear to initiate the cell cycle. Cyclins appear to trigger the cell‘s entry into the cell cycle and into mitosis by activating the transcription of c-myc and c-mos. Proteins from c-myc and c-mos also reside in the nucleus and form complexes that stimulate DNA synthesis. Other factors enter from the cytoplasm (e.g., ras, myc); these are transcribed and activated by cytoplasmic kinases (see step 3). Several normal products of proto-oncogene kinases in the cytoplasm also activate gene transcription, particularly PKC, which acts directly as a DNA promoter and stimulates the production of c-jun and c-fos proteins; this junfos complex binds to specific promoter regions of DNA and stimulates DNA transcription.

Example: c-myc oncogene (Others: jun, fos, erb-A, rel, ski, myb)

a. Normal function. The c-myc are a family of proteins with direct nuclear regulatory activity. The presence of active myc appears necessary for cell division. Dimethylsulfoxide inhibits myc transcription, stops cell division, and can cause differentiation of leukemic blast cells in culture. High levels of c-myc are found in immature blood cells and gastro-intestinal cells. Both c-myc and the c-junc-fos protein products form dimers connected at a leucine-rich region called the «leucine zipper» near the site of attachment to promoter genes.

b. Abnormalities in cancer cells. Amplification of the myc gene can be so prolific that the multiple copies sometimes cannot remain on a chromosome. Extrachromosomal «double minutes» often microscopically give visible evidence of such amplification, with high quantities of active gene product being formed.

c. Associated human cancers include Burkitt’s lymphoma, B-cell lymphomas, promyelocytic leukemia, neuroblastoma, small cell lung cancer, and colon cancer.

d. Effect of abnormalities on prognosis. In neuroblastoma the prognosis is poorer in direct proportion to levels of N -myc (N = neuroblastoma) for this member of the myc gene family.

Step 5. Growth inhibitor genes produce substances, typically phosphatases, which normally inactivate growth-promoting oncogene products and inhibit cell proliferation. Mutations that make these genes nonfunctional lead to uncontrolled cell proliferation and malignancy. Examples of these mutations and their associated tumors include RB1 (retinoblastoma and some osteosarcomas), WT1 (Wilms‘ tumor), NF1 (neurofibromatosis), MEN1 (multiple endocrine neoplasia), and FAP1 (familial adenomatous polyposis of the colon).

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