Cell Biology (Notes 37)

Final Exam

What is an overview of carcinogenesis?

• There are many ways to introduce mutations to DNA

  • Radiation

  • Chemicals

  • Infectious agents

  • Heredity

• Those mutations will convert proto-oncogenes to oncogenes, and inactivates tumor suppressor genes

• This leads to the instability of the genome

What is self-sufficiency?

• Proliferation in the absence of growth factors

  • This phenomenon allows cancer cells to grow and divide independently, bypassing normal regulatory mechanisms that require external signals for cell growth

• The graph:

  • X-axis: days of time in culture

  • Y-axis: cell number

  • The expectation is that the longer cells are in culture, the more they’ll grow

  • There are four lines on this graph:

    • Normal cells + serum growth factors: after blood clots, platelets in the blood release growth factors

      • Growth factors are important for the proliferation for the cells in culture

      • Exponential rise and then plateau

    • Normal cells - serum growth factors: a little growth, then plateau

    • Cancer cells + serum growth factors: more robust growth and division - continuous line/growth (exponential)

    • Cancer cells - serum growth factors: straight line

      • Cancer cells are able to growth in the absence of growth factors

      • Goes into a story of cell signaling

• The signaling story:

  • Signal molecule causes receptor tyrosine kinase to autophosphorylate

  • Adaptor protein activates Ras activating protein, which activates Ras to be GTP bound

  • The onward signaling continues the cell cycle

  • All of this signaling is what you need to get the cell past the regulating events and get it to S phase

  • Cancer cells don’t pay attention to the regulating

  • Presence of growth factor regulates … cancer cells signal in the absence of growth factor

What are two examples of self-sufficiency?

• Her2 receptor is mutated in about 30-35% of breast cancers:

  • Epidermal receptor that binds growth factor

  • In cancer, the protein being expressed by the proton-oncogene will develop a mutation that will lead to an amino acid substitution (oncogenic mutation)

    • Valine —> Glutamine

  • The mutation is in the transmembrane domain of the receptor spanning the plasma membrane

  • That mutation is sufficient to change the structure of the protein thus the receptors will auto-phosphorylate themselves in the complete absence of a growth factor

  • Once the mutation is acquired, Her2 becomes Neu, which is an oncoprotein

• EGF receptor is a tyrosine kinase or a mutation that the protein that’s expressed in the cancer cell is a truncated protein, it lacks the extracellular domain —> no capacity to bind a growth factor—> renamed ErbB

  • Because it lacks an extracellular domain, it’ll go through a conformational change to allow the intracellular domain to undergo the process of autophosphorylation

What is insensitivity?

• Insensitivity - antigrowth signals no longer recognized which results in release of G1 Arrest and degradation of ECM

• There is a gene called p15 that encodes an inhibitor of cell cycle progression (this is a NORMAL cell)

  • If you are lacking p15, you’re going to continually go through cell divisions

• There is another gene called PAI-1, which encodes an inhibitor of a protease that is needed to destroy the extracellular matrix

  • To keep the ECM intact, we can express an inhibitor of a destructive protease

• In a normal cell, both genes must be expressed

  • Smad protein complex enters the nucleus and interacts with other promoters/transcription factors to regulate the two genes (p15 and PAI-1)

  • Smad becomes activated in response to receptors that are activated on the cell surface

    • These receptors are serine/threonine kinase receptors that can become phosphorylated

    • Those phosphorylation events are occurring in the intracellular domain

What is transforming growth factor beta (TGF beta)?

• Growth factor that ensures the cells are NOT transformed

• If there are mutations that interrupt this signaling event, the cell will start acquiring the characteristics of a cancer cell

• A cell acquires mutations that prevents this signal from occurring - release of arrest in G1, cells enter the cell cycle, degradation of the ECM

What do cells need to do to stay healthy?

• Have contact with the extracellular matrix

• Experiment:

  • Placed glass squares of different dimensions in a medium and covered them in extracellular matrix proteins

    • On each protein, they placed a cell

  • On a graph:

    • X-axis - adhesive island area

    • Y-axis - apoptosis on the left

    • Y-axis - DNA synthesis on the right

  • Over time, the squares would experience higher amounts of DNA synthesis (cells)

  • Over time, the cells would experience lower apoptosis

How do you measure DNA synthesis?

• Radioactive labeling

• DNA probes

• Measures increase or decrease of DNA content

How do you measure apoptosis?

• Measure markers of apoptosis such as caspase activation, DNA fragmentation, and changes in mitochondrial membrane potential

What did they find from the experiment?

• On the smaller adhesive islands, cells showed increased apoptosis and decreased DNA synthesis compared to larger islands, indicating that cell adhesion area directly influences cell survival and proliferation

• cells require sufficient contact with the extracellular matrix for optimal function. Larger adhesive surfaces promote cell survival and growth, while smaller areas lead to increased cell death

• On the smallest island, there is barely any cell growing

• The conclusion of the experiment is that the cell’s contact with the ECM is a matter of life and death

  • If there is not sufficient contact with extracellular matrix, cell will undergo process of programmed cell death

Anoikis

• Programmed cell death that is triggered by lack of contact with extracellular matrix

• Important for excavation of tissues and prevention of tumors

What do the colors mean?

• Red (ECM) - laminin

• Green (apoptosis) - caspase

• Blue - nuclei

What does each cell look like during anoikis?

• On the right: the cell has no caspase (green):

  • The cells in the center are not dying - they’re going to keep multiplying

    • Lack of green (caspase)

    • Breast tumor

    • Bcl-2 levels are extremely high

      • With that, there is a potent inhibitor of programmed cell death

      • This is an escape from apoptosis (blocking apoptosis)

• On the left: the cell has caspase (green):

  • The cells in the center are dying - not multiplying

    • Lack of green

    • Normal level of Bcl-2, which is putting a brake on programmed cell death

• This is all happening in human mammary epithelial cells

What is evasion (escape of apoptosis)?

• If there are high levels of MDM2, p53, a tumor suppressor gene, does not become activated

  • There is no expression of proteins needed for cell death

  • Expression of proteins at high levels can prevent cells from dying

What is immortality?

• Limitless replicative potential

• Stem cells can keep REPLICATING INDEFINITELY if the conditions are correct

• The problem with cancer is that ALL THE CELLS have acquired immortality or a limitless replicative potential

• These are all processes that are uncoupling a cell from mechanisms that are limiting cell proliferation — cell is uncoupled from natural process of programmed cell death

  • These uncoupling events do not ensure limitless replicative potential —> something else is happening

What does cancer-like immortality mean?

• No limit as to how many times it can divide

Process of Immortality

• While self-sufficiency, insensitivity, and evasion uncouple cells from mechanisms that limit proliferation but does not ensure LIMITLESS proliferation

• Immortality allows limitless proliferation… but what prevents limitless proliferation?

Cell Lineages Undergo Replicative Senescence and Crisis

• Has gone into G0 in cell cycle and is resting, but it has full potential to re-enter the cell cycle and go through more rounds of cell division

• The graph:

  • X-axis: time (days after initiation of culture)

  • Y-axis: growth rate of culture

  • Certain cells experience a dip in growth rate - those cells are SENESCENT

    • Never re-enter the cell cycle

    • No growth happening in the culture

  • People look at the REPLICATIVE AGE of a cell (how many cell divisions has a cell gone through - each division is a birthday)

    • Hayflick said that when cell has gone through max number of divisions and gone into terminal G0 state, the cell has reached replicative limit

    • This is called the Hayflick Limit

  • These cells have a stable karyotype - the chromosomes are fine - the cell just won’t replicate

What drives cell senescence?

• Metabolic stress on the physiology of the cell (accumulation of Reactive Oxygen Species - ROS)

  • ROS is causing molecular damage on the cell

  • In total, the damage is sensed by the cell and the cell isn’t moving forward anymore

What are the differences between normal and senescent cells?

• Senescent cells are wider and more stretched out

What are crisis cells?

• Defined by the VIABILITY of a cell

• Viability of the culture CRASHES when cells are in crisis

• Crisis is DIFFERENT from senescence

  • Crisis is where you have a lot of extreme instability of the genome

  • Unstable karyotypes - chromosomes are breaking and fusing together

  • Odd chromosomal morphologies - driven by the loss of the ends of the chromosomes or the telomeres

  • These cells will start dying and the loss of viability leads to apoptosis - the cell has damage that it cannot repair

There are many ways for a cell to die

• Leading cause of cell death: apoptosis

• Mitotic catastrophe - where chromosomes can’t segregate properly

What are the basics of DNA replication?

• Origins of replication

  • Leading strands

  • Lagging strands - Okazaki fragments

• There needs to be a primer for which DNA synthesis begins - that primer is a molecule of RNA that is annealing to the template strand

  • The polymerase recognizes the RNA primer and uses the template strand of DNA to synthesize the complementary strand of DNA

  • The RNA primer has to proceed where DNA synthesis will begin

• However, what happens at the end of the chromosome?

Telomeres

• Primer is not covering the ends of the chromosome in the gap

  • Another primer is needed at the end of the gap to fill in the gap

  • After each round of replication, you’re always left with a gap at the end of the chromosome

    • Thus, the chromosome gets shorter and shorter with each generation

What happens to telomeres?

• Ends of chromosomes are in red

• As the ends of the chromosome shorten, the expression of certain genes are lost because they’re no longer there or half there

  • Those are the cells that are entering crisis because they no longer have a full complement of genes

• With each generation, the telomeric DNA becomes shorter and shorter and lose the parts of the chromosome that have coding sequences

  • These cells enter crisis

How do the ends of chromosomes maintain their structure?

• Ends of the chromosomes that are not functional - repetitive sequence

• How do they produce telomeres in the absence of an RNA primer to allow DNA synthesis to synthesize the ends of the chromosomes?

  • The cell expresses telomerase

Telomerase

• Binds a short RNA molecule (repetitive A’s and C’s)

• That molecule serves as a template

• That RNA gives information to the cell such that the telomerase can insert these corresponding complementary nucleotides (where there’s a C, there’s G at the 3’ end)

  • Telomerase is synthesizing the end of the chromosome and giving it the extra repetitive sequence

• There’s still a gap - extend template strand

  • To synthesize missing DNA, all the normal methods of DNA replication occur - priming, DNA polymerase to fill in the gap

  • The telomeric sequences are repetitive, but they have this interesting ability to fold back on themselves and form a loop

  • Both ends of the chromosome have a loop of DNA - that’s how you finish the end of the chromosome

  • With each cell generation that these chromosomal ends become shorter and shorter and the cells enter crisis

• What does this mean for the life of the cell and why does cell enter crisis?

Barbara McClintock

• Through her work with maize genetics, she deduced that there is chromosomal breakage and fusion events occurring

  • Two broken chromosomes fuse together to form a new chromosome

  • This is especially important at the ends of the chromosomes

  • Fluorescent red chromosomes with the green dots at the end are visualized with FISH

    • Nucleic acid is used as a probe attached to another molecule

    • Every chromosome has a green dot - telomeric DNA is located here

    • Over each generation, the telomeres become shorter - they are formed early in life and telomerase shuts off —> ends of chromosome

    • With each cell division, chromosome ends become shorter

    • Chromosomes become too shorter —> crisis

• Chromosomal instability drives crisis

  • Telomeres shown in red

  • Erode telomeres with each cell division - unprotected chromosomal ends

  • Those ends can fuse end to end — homologous chromosomes and sister chromatids and the ends are eroded

    • Arms will fuse with each other

Where does the genetic instability come in?

• Fused chromosomes in anaphase

• Those kinetochores are the mitotic spindle — tension is being put on that odd chromosome

  • Because of the tension, there’s a short chromosome and a longer chromosome with an exposed end

  • Along comes a non-homologous chromosome —> brown chromosome will fuse with the blue chromosome

  • Next mitosis - new breakage

• Eventually, the karyotype of the chromosomes will be a tangled mess of chromosomes

• All of these bits of chromosomes are attached to each other

  • This is where the mechanism of programmed cell death kicks in and the cell dies from apoptosis

What is chronic myelogeneous leukemia?

• Telomeric DNA is still there - but there is a breakage of chromosomes that leads to the development of cancer

  • Chromsomes 9 and 12 break and fuse

    • On Chromosome 9 - there’s ABL

    • On Chromsome 22 - there’s BCR

  • Breakage occurs about halfway through each gene - when two chromosomal fragments fuse together to form this small chromosome (Philadelphia chromosome)

    • Part of the BCR and the ABL gene are together - creating a chimeric gene

• People don’t know WHY this leads to a cancerous state — ABL encodes a kinase and BCR encodes a GEF

• We know this odd protein is highly expressed in cancer cells

• Chromosomal translocations lead to the formation of novel fusion proteins that are hyperactive

  • This is a case where you have a proto-oncogene becoming an oncogene

What is Burkett’s lymphoma?

• Chromosomal translocation results in active region of chromosome 14 in close proximity to the MYC gene of chromosome 8

• These two chromosomes are translocating pieces —> leads to excess Myc protein

• Myc is one of the early transcription factors needed for entering S phase - in excess, Myc will push cells into S phase

How do cancer cells escape crisis?

• They escape crisis by expressing TELOMERASE (enzyme that builds the ends of the chromosomes)

• Blackburn, Greider, Szostak found this discovery in a protist; McClintock also won a Nobel Prize on chromosome stability

• The experiment:

  • X-axis: time (days)

  • Y-axis: culture growth (PDs)

  • At a certain point, growth of cell culture stops

    • HEK cells without hTERT has entered crisis

    • HEK cells with hTERT (telomerase) keeps increasing and increasing

  • Expressing telomerase is sufficient for allowing a cell to escape crisis

  • This is what is happening in cancer cells and allows them to keep replicating limitlessly

How does loss of telomerase trigger crisis and inhibit neoplastic growth?

• There are four different cancer cell lines

  • Three lines per graph

  • Time (days) vs culture growth (PD)

    • Green lines show wild type level of telomerase (hTERT)

    • Blue line is the control

    • The red line is the mutated hTERT

  • Line functioning telomerase undergo VERY FEW DOUBLINGS

  • The expression of telomerase is ABSOLUTELY NECESSARY for the doubling of the cell population

  • Loss of telomerase triggers crisis and inhibits neoplastic and cancerous growth of cells

Summarizing Telomeres

• Somatic cells have reduced telomeric DNA sequences because with each cell division, you lose a little bit more of the chromosome

• Eventually, once you lose your last telomeric DNA sequence, those cells enter crisis

• Cancer cells expressing telomerase have very long telomeres - those cells will keep growing over time, yet those cells can display genetic instabilities where there are breakages and translocations on the length of chromosomes

  • Not at end because ends are protected

• Stem cells also have long telomeres so they can divide limitlessly

• Embryonic cells also have long telomeres, the starting point of our DNA

  • Why do we have long telomeres in embryonic but not somatic cells?

    • Because adult cells are NOT expressing telomerase

    • But embryonic cells are expressing telomerase because they are building telomeres to generate a population of 37 trillion cells