finite cell lines can be converted to infinite cell lines through transformation

1. IN VITRO TRANSFORMATION
MADE BY:
VAISHALI JAIN
M.Sc. (Prev.) MEDICAL
BIOTECHNOLOGY

2. INTRODUCTION
• The alteration in a culture that gives rise to a continuous cell line is commonly called in vitro
transformation .
• Transfection-Artificial introduction of DNA or DNA transfer into mammalian cells
• Transformation is associated with genetic instability and three major classes of phenotype change,
one or all which may be expressed :
• Immortalization – the acquisition of an infinite life span
• Aberrant growth control – the loss of contact inhibition of cell motility, density, limitation of cell
proliferation and anchorage dependence
• Malignancy – Growth of invasive tumors

3. • Transformation of cells may occur due to any one of the following
causes that ultimately result in a changed genetic material:
• i. Spontaneous.
• ii. Infection with transforming virus.
• iii. From gene transfection.
• iv. Exposure to chemical carcinogens.
• v. Exposure to ionizing radiations.

4. GENETIC INSTABILITY
• Human finite cell lines are stable
• Mouse cell lines are genetically unstable and transform easily
• Continuous cell lines (tumorigenic) of all species are unstable
• Two causes of genetic heterogeneity
- Spontaneous mutation is higher in vitro
- Mutant cells cannot be eliminated without impairment in their growth
capacity

5. CHROMOSOMAL ABERRATIONS

6. IMMORTALIZATION
• Most normal cells have a finite life span of 20 to 100 generations
• Rodent cells can produce continuous cell lines with an infinite life span
• Rodent cells are karyotypically normal at isolation and undergo changes after 12
th generation
• If maintained at a low cell density and not allowed to remain at confluence for
any length of time, they remain sensitive to contact inhibition and density
limitation of growth
• If allowed to remain at confluence for extended periods – reduced contact
inhibition- cells pile up – overgrowth- will be seen in subsequent subcultures -
tumorigenic

8. CONTROL OF SENESCENCE
• Senescence genes in finite cells negatively control cell cycle
progression
• Immortalization involves both inactivation of senescence and cell
cycle regulatory genes such as Rb and p53
• Product of SV40 LT gene – T antigen binds to Rb and p53
• Allows extended proliferative life span and restricts DNA
surveillance activity of genes
- Increases genomic instability
- Increases chances of mutations

9. • Immortalization – does not imply development of aberrant growth
control and malignancy
- Retains contact inhibition of cell motility, density limitation of cell
proliferation and anchorage dependence and are non-tumorigenic
- Some aspects of growth control are abnormal and likely increase of
genomic instability

10. • Mammalian cells transfected or retrovirally infected with
immortalizing gene before they enter senescence
• Extends proliferative life span for 20-30 population doublings
- Cells cease proliferation and enter crisis
- Up to several months in crisis – subset of immortal cells overgrows
- Fraction of immortal cells obtained

11. Telomerase-Induced Immortalization
• Telomerase or terminal transferase – composed of two subunits –
RNA component (hTR) and a protein catalytic subunit (hTERT)
• hTR is expressed in normal and malignant tissues
• hTERT is expressed in tumors, germ cell lines and activated
lymphocytes
• Transfecting cells with hTERT extends life span of cell line
- Some cells become immortal but not malignantly transformed

14. ABERRANT GROWTH CONTROL
• Cells cultured from tumors ,as well as cultures that have transformed
in vitro show aberrations in growth control such as growth to higher
saturation densities , clonogenicity in agar and growth on confluent
monolayers of homologous cells.
• These cell lines exhibit lower serum or growth factor dependence,
usually form clones with a higher saturation efficiency and are
assumed to have some degree of autonomous growth control by
overexpression of oncogenes or by deletion of suppressor genes.
• Growth control is often autocrine.

15. ANCHORAGE INDEPENDENCE
• Transformation due to cell surface modifications in cell surface
glycoproteins and adhesion molecules
• Fibronectin or large extracellular transformation sensitive (LETS)
protein is lost from surface of transformed fibroblasts due to
alteration in integrins. This contributes to decrease in cell-cell and
cell-substrate adhesions and to decrease requirement for attachment.
• Transform cells may lack specific CAMs.

16. CONTACT INHIBITION
• Detected morphologically by the formation of a disoriented
monolayer of cells or rounded cells in foci.

17. SERUM DEPENDENCE
• Transformed cells have a lower serum dependence due to secretion
of autocrine growth factors.
• Cause nontransformed cells to adopt transformed phenotype and
grow in suspension
• Produce interleukins 1,2 and 3 along with colony stimulating
factors (CSF)
• Hormones released

18. Aberrant growth control by Oncogenes
• Overexpression of oncogenes
• Modified receptors erb-B2 oncogene product, modified G protein
and overexpression of genes regulating stages in signal transduction
(src kinase) or transcriptional control (myc, fos and jun)
• It is permanently active and cannot be easily regulated

19. TUMORIGENICITY
• Transformation culminates into neoplastic cells
• Malignant tumors – increased growth rate, reduced anchorage
dependence, more pronounced aneuploidy and immortalization
• All cell lineages present within a tumor need not have same
transformed properties
• Same properties cannot be expressed in every tumor

20. Tumorigenicity by malignancy
• Cells have developed the capacity to generate invasive tumors if
implanted in vivo into an isologous host or if transplanted as a
xenograft into an immune-deprived animal.

21. INVASIVENESS

23. Tumorigenicity by angiogenesis
• Tumor cells release factors – VEGF, EGF-2 and angiogenin –
capable of neovascularization
• Tumor cells produce proteolytic enzymes – plasminogen activator –
helps the tumor cells in not attaching to any surface – help them
invading other cells