This document discusses in vitro transformation, which is the alteration of cells in culture that results in a continuous cell line. In vitro transformation can occur spontaneously or be induced by viruses, transfection, carcinogens, or radiation. Transformed cells exhibit immortalization, aberrant growth control, and malignancy. Immortalization involves infinite lifespan and loss of contact inhibition and density-dependent growth control. Aberrant growth control includes loss of serum dependence and anchorage independence. Malignancy is characterized by tumorigenicity, invasiveness, angiogenesis, and metastasis.

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