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NEOPLASTIC TRANSFORMATION IN VITRO



How to study the early stages of carcinogenesis in the lab:
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Properties of neoplastic and immortalized cells in culture
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Fundamental aspects of animal cell culture:
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Defined media and nutrients:
• Minimal Essential Medium (MEM)
• 13 essential amino acids (L-glutamine)
• 8 vitamins
• Inorganic salts and glucose

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, but it’s main function is to supply growth factors
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Serum can sometimes be reduced or replaced by defined factors but
cannot be eliminated altogether for normal cells
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EGF, FGF and insulin may favour growth of fibroblasts
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Specialized cell types require specific growth factors, for example,
keratinocytes need FGF, endothelial cells need FGF
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g
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Cell-to-cell contact inhibition:
• Sparse cells are highly motile in culture
• Cell collision inhibits forward movement
• Cell contact causes regular alignment and patterning of cells
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Density inhibition: confluent cells enter G0/G1 of the cell cycle and
arrest their growth
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Anchorage dependent:
• Most normal cells cannot grow in suspension (agar, agarose,
methylcellulose)
• Fibroblasts can survive, attach, grow and spread on suitable
substrates (E
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plastic, glass)

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Epithelial and endothelial cells undergo programmed cell death in
suspension – apoptosis
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Non-tumorigenic:
• Culturing cells per se does not necessarily lead to neoplastic
transformation
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5
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It states that a normal human
cell can only replicate and divide forty to sixty times before it
cannot divide anymore, and will break down by programmed cell
death or apoptosis
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g
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• Cell senescence may be an anti-tumour mechanism to prevent
run-away growth
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Rodent cell cultures occasionally pass through senescence and resume
rapid growth
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Such cells are called immortal – immortalization generally correlates
with gross alterations in karyotype (chromosome number, heteroploid or
aneuploidy)
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Spontaneous immortalization is frequent with rodent cells but rare with
human cells
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Immortalized cells are sometimes normal – content and density
inhibition, anchorage-dependence and lack tumourigencity, and differ
from normal cells only in terms of life-span
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Established cell lines provide useful models, e
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mouse embryo
fibroblasts
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Certain viruses can cause cancer in animals
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Altered growth properties often due to reduced growth factor
dependence: transformed cells grow in low serum (<10%) and behave
as though constitutively activated by growth factors
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Properties of transformed cells:
• Altered metabolism – increased reliance on glycolysis
• Altered membrane properties – elevated nutrient transport,
excessive blebbing, increased motility of membrane protein
(increased membrane fluidity)
• Reduced substrate adhesion – more rounded shape


Growth factor dependence:
1
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Density inhibition:
• Mainly due to exhaustion of growth factors
• Saturation density is proportional to the serum concentration
• Add more serum to dense cultures à growth resumes
• Transformed cells reach a higher density because they need
less serum

Anchorage Dependence:
1
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e
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Senescence:
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”
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13
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This is a cancer hallmark that may allow some therapeutic intervention
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• Chemically modified oligonucleotide that inhibits the interaction
between telomerase and telomeric DNA, thereby blocking cancer
cell proliferation and xenograft growth in mice
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Mechanism of growth arrest:
1
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2
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• Stress-induced senescence via p16 induction
• Oncogene-induced premature senescence causing p53-p21
induction and p16 expression
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However, telomere loss may not be a universal tumour suppressing
mechanism or cause of cell senescence:
• Mouse cells have longer telomeres than human cells but have a
much shorter life span in culture
• Telomerase knockout mice also remain viable for about 4-5
generations
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• Human epithelial cells have two pathways to cell senescence
whereas mouse cells only have one pathway in response to cell
stress
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Cells that have inactivated both the p53 and Rb patwhays lack the
mechanisms for inducting growth arrest
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Such cells continue to proliferate, causing progressive telomere
shortening
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The eventual loss of the protective telomere cap leads to crisis:
• Chromosome fusion,
• Mitotic abnormalities
• Extensive cell death (Apoptosis)
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The rare cells that do escape crisis generally have reactivated telomerase
expression but only after the genome is massively scrambled
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Mouse cells emerge from crisis much more readily than human cells
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Also, human cells have a much more efficient repair mechanisms in place
to suppress transformation, in that, a life span of a mouse is about 2 and a
half years, in comparison to 75 years for a human
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The hallmarks of cancer include:
• Self-sufficiency in growth signals – growth autonomy
• Insensitivity to anti-growth signals – cell differentiation, tumour
suppressor pathways
• Evasion of apoptosis
• Limitless replicative potential – avoidance of cell senescence
• Sustained angiogenesis
• Tissue invasion and metastasis







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