The Discovery of Oncogene

曲颖（ 1040800020 ）

What is the function of the src gene product?

How does src alter the behavior of normal cell that already possesses a copy of the cellular gene?

Identification of the product of the src gene

Prepare the antibodies from RSV-infected animals

Precipitate( 沉淀 ) the protein from extracts of transformed cells by antibodies

Synthesize the protein using the isolated viral gene as a template in a cell-free protein-synthesizing system

A protein of 60,000 daltons—pp60src

When pp60src was incubated with [32P]ATP, radioactive phosphate groups were transferred to the heavy chains of the associated antibody molecules used in the immunoprecipitation( 免疫共沉淀 ).

src gene codes for an enzyme that possesses protein kinase activity.

Function of the product of the src gene

Cells infected with ASV were fixed, sectioned, and incubated with ferritin( 铁蛋白 )-labeled antibodies against pp60src, the antibodies were found to be localized on the inner surface of the plasma membrane,

Orientation of the product of the src gene

suggesting a concentration of the src gene product in this part of the cell.

A protein of 60,000 daltons—pp60src

src gene product:

An enzyme that possesses protein kinase activity.

Concentrate on the inner surface of the plasma membrane

What is the function of the scr gene product?

The number of phosphorylated tyrosine residues in proteins of RSV-transformed cells was approximately eight times higher than that of control cells.

Viral vesion of the gene may induce transformation because it functions at a higher level of activity than the cellular version.

＊ An increased activity of an oncogene product could be a key to converting a normal cell into a malignant cell .

＊ Malignant cell could also be induced by an oncogene that contained an altered nucleotide sequence.

How does src alter the behavior of normal cell that already possesses a copy of the cellular gene?

Obtain 15 different malignant cell lines that were derived from mouse cells that had been treated with a carcinogenic( 致癌的 ) chemical. (Made malignant without exposing them to viruses .)

DNA from each of these cell lines was extracted

Transfect a type of nonmalignant mouse fibroblast called an NIH3T3 cell.

The fibroblasts were grown in vitro.

The cultrues were screened for the formation of clumps(foci) that contained cells that had been transformed by the added DNA.

DNA transfection

Demonstration ofOncogenes by Gene Transfer DNA, isolated from tumor cells (growing in culture), is mixed with calcium phosphate and added to normal mouse cells under conditions where the DNA can enter the cells (this process is called transfection). If an oncogene is present in the DNA, a small number of the normal cells will become oncogenically transformed (i.e. “tumor-like”). If these transformed cells are isolated and injected into a mouse, they grow to form a tumor.

Five of the fifteen cell lines tested yielded DNA that could transform the recipient NIH3T3 cells (DNA from normal cells lacked this capability) .

Carcinogenic chemicals produced alterations in the nucleotide sequences of genes that gave the altered genes the ability to transform other cells.

DNA isolated from human tumor cells can transform mouse NIH3T3 cells following transfection.

No evidence of viral DNA was detected in these cells.

Some human cancer cells contain an activated oncogene that can be transmitted to other cells ,causing their transformation.

Attention changed to human cancer

Three different laboratories reported the same isolation and cloning of an unidentified gene from human bladder carcinoma ( 膀胱癌 ) cells that can transform mouse NIH3T3 fibroblasts （成纤维细胞）—— named ras that is carried by the Harvey sarcoma virus, which is a rat RNA tumor virus.

Nucleotide sequence analysis indicated that the DNA from the malignant bladder cells is activated as a result of a single base substitution within the coding region of the gene.

A guanine(G)-containing nucleotide at a specific site in the DNA of the proto-oncogene had been converted to a thymidine(T) in the activated oncogene.

This base substitution results in the replacement of a valine( 缬氨酸 ) for a glycine （甘氨酸） as the twelfth amino acid residue of the polypeptide.

Thus RAS can be activated to induce transformation by two totally different pathways: either by increasing its expression or by altering the amino acid sequence of its encoded polypeptide.

The change in the viral gene substitutes an arginine for the normal glycine. It was apparent that this particular glycine residue plays a critical role in the structures and function of this protein.

Both the RAS gene and SRC gene are proto-oncogenes.

The Biological Role of Protooncogene Products

The discovery of protooncogenes in normal cells raises questions regarding their role in normal cellular processes.

Are protooncogenes silently sitting within normal cells “waiting” to cause cancer, or do these genes play a role in normal physiological processes?

Research shows that the protooncogene and its product plays key role in some physiological process. The processes in which protooncogenes are involved is quite varied; however, most protooncogenes have been found to be involved in the mechanisms that govern the growth and differentiation of cells. The biochemical function of protooncogene products is even more varied.

The expression of oncogenes (literally “cancer genes”) within cells is a crucial event in the early stages of tumor formation. Oncogenes can arise in cells via two mechanisms: infection of cells by tumor viruses and conversion (mutation) of cellular protooncogenes to oncogenes. These discoveries have important implications for the prevention, detection and treatment of cancer.

That three decades of intensive research in oncology and related basic sciences has not led to a cure for cancer may seem disappointing to the general public; it has, however, led scientists to a much better understanding of the ‘problem’ of cancer. The discovery of the genetic basis for tumorigenesis, along with the advent of ‘biotechnology’ holds great promise that the next thirty years will bring both more effective anti-tumor therapies and greatly improved diagnosis of tumors.

We believe that one day we will defeat cancer

eventually !

1-9-2007