Transformation of Animal Cells

TRANSFORMATION OF CELLS

It’s the change in the phenotype of a cell due to a new genetic material. As regards the culture cells, transformation involves spontaneous or induced permanent phenotypic alteration as a result of heritable changes in DNA and consequently genes expression.

Transformation of cells may occur due to any one of the following causes result in a changed genetic material:

1.      Spontaneous

2.      Infection with transforming virus

3.      From gene transfection

4.      Exposure to chemical carcinogen’s

5.      Exposure to ionizing radiations

Transformation is associated with genetic instability, immortalization, aberrant growth controle and malignancy

Genetic instability: In general, the cell line in culture is prone to genetic instability. A majority of normal finite cell lines are usually genetically unstable, and can get easily transformed. The continuous cell lines derived from tumors of all species are unstable. The normal occurring genetic variation in the cultured cells are due to the following causes:

1.      High rate of spontaneous mutation in the invitro condition, possibly due to high rate of cell proliferation.

2.      The continued presence of mutant cells in the culture, as they are not normally eliminated.

Immortalization: The finite life span of cultured cells is regulated by about 10 senescence genes. Those dominantly acting genes synthesize products which inhibit the cell cycle progression. Its strongly believed that immortalization occurs due to inactivation of some of the cell cycle regulatory genes. E,g, Retinoblastoma, p53 genes.

Immortalization of cells by viral genes: several viral genes can be used to immortalize cells. Some of these genes are listed below:

·         SV40LT

·         HPV16E6/E7

·         hTRT

·         EBV

Among the above viral genes, SV40LT is most commonly used to induce immortalization. The product of this gene (T antigen) bind to senescence gene such as Rb and p53. This binding restricts surveillance activity of senescence genes. The result is an increased genomic instability and activity, leading to further mutations favoring immortalization.

For the process of immortalization, the cells are infected with retrovirus containing immortalizing gene before they enter senescence. By this way, the life span of the cells can be extended by 20-30 population doublings. Thereafter the cell cease to proliferate, and enter a crisis phase that may lost for several months. At the end of the crisis phase, a small portion of cells can be growing, and eventually become immortalized.

Immortalization of cells by telomerase induction: The most cause of finite life span of cells is due to telomeric shortening, followed by cell death. If the cells are transfected with telomerase gene htrt, the life span of the cells can be extended, and a small portion of these cells become immortal.

Aberrant growth control: the transformed cells and the cells from tumors grown in culture show many aberration with respect to growth and its control. The growth characteristics of these cells are listed as:

Genetic characters’	Structural characters’	Growth characters’	Neoplastic characters’
Aneuploid	Altered cytoskeleton	Immortalized cells	Tumorigenic
Heteroploid	Changed extracellular matrix	Loss of contact inhibition	Invasive
High spontaneous mutation rate.	Modified expression of cells adhesion molecules	Anchorage independent	Increased protease secretion
Over expressed oncogenes	Disrupted cell polarity	Density limitation of growth reduced
Mutated or deleted suppressor genes		Growth factor independent
		Low serum requirement
		Shorter population doubling time

Anchorage independence

There occur several changes on the cell surface of transformed cells. These include alteration in the cell surface glycoprotein and integrins, and loss of fibronectin. Some of the transformed cells may totally lack cell adhesion molecules. The modification on the surface of transformed calls leads to a decrease in call-cell and cell-substrate adhesion. The net result is that there is a reduced requirement for attachment and spreading of the cells to proliferate. This phenomenon is reffered to as “anchorage independence”

Anchorage independent cells grow in a disorganized fashion. These calls may be comparable with the tumor cell detached from the native tissue which can grow in foreign tissues, e.g the spread of cancer from one part of body to another.

Contact inhibition:

The transformed cells are characterized by loss of contact inhibition. This can be observed by the morphological changes in the disoriented and disorganized monolayer cells. This result in a reduced density limitation of growth, consequently leading to higher saturation density compared to normal cells.

Low serum requirement:

In general, transformed cells or tumor cells have lower serum dependence than the normal cells. This is mostly due to the secretion of autocrine growth factors by the transformed cells. Some growth factors produced by tumor cells are:

·         Colony stimulating factor (CSF)

·         Transforming growth factor (TGFa)

·         Interleukin I, II, and III

·         Vasoactive intestinal peptide (VIP)

·         Gastrin release peptide.

Tumorigenicity:

Cell transformation is a complex process that often results in the formation of neoplastic cells. The cell lines obtained from malignant tumors are already transformed. Such cells may undergo further transformation in the invitro culture due to

·         Increased growth rate

·         Immortalization

·         Reduced anchorage dependence.