Exam IV: Suppression of Cancer Cells Article

Question 1

What are 4 advantages of using Adeno-Associated virus (AAV)?

Answer 1

1. Non-immunogenic
2. Low pathogenicity
3. Long-term gene expression
4. Wide tissue or cell tropism

Question 2

What is the disadvantage of AAV?

Answer 2

Lack of tissue specificity

Question 3

What was the overall plan of this paper?

Answer 3

Modify the tropism of AAV capsids by inserting the hTERT promoter in front of IFNbeta gene and putting the whole thing into the capsid. IFNbeta will then only be activated in cancer cells/germ cells that have telomerase on

Question 4

What does telomerase do? What cells is it expressed in (3)?

Answer 4

Maintains the length of telomeres by adding G repeated
Expressed in gametes, stem, and tumor cells

Question 5

What are the 4 benefits of IFNb?

Answer 5

1. Upregulation of MHC class I expression
2. Activation of NK cells and cytotoxic T lymphocytes
3. Inhibition of tumor angiogenesis
4. Induction of tumor cell apoptosis

Question 6

What are the 2 disadvantages to IFNb?

Answer 6

1. Short half life
2. Systematic toxicity at high doses

Question 7

Explain the image

Answer 7

Testing to see if the tumor specific targeting is working

ELISA assay measuring IFNb concentration

Higher amounts of IFNb in tumor cell lines

Question 8

Explain the image

Answer 8

Testing to see if the IFNb expression will cause tumor-specific killing

Saw no death in normal cells, cell death in tumor cells

All cells are infected with AAV = but toxicity is low enough

Question 9

Explain the image

Answer 9

Tested to see how the IFNb was killing the cells

It is mediated by apoptosis

They know this because they stained for apoptotic cells in light blue

Question 10

Explain the image

Answer 10

Tested to see if that worked in vivo

Looked at the amount of tumor loss in mice colorectal tumors

Looked at overall survivability in mice after treatment

Question 11

Explain the image

Answer 11

HE staining = tumor vascularity. IFNb decreased tumor vasculrity

IHC: expression of IFNb (using antibodies to produce a dark dye)

TUNEL: TDT binds to broken DNA caused by apoptosis > brown apperance

TDT adds a labeled dinucleotide to the exposed OH group

Question 12

Explain the image

Answer 12

Flow cytometry assay

Each cell depending on the amount of DNA will fourece at different levels

the peak for apoptosis is shifted forward

Question 13

Explain the image

Answer 13

Western Blot looking at the proteins involved in the intrinsic mitochondrial apoptosis pathway

Question 14

Explain the image

Answer 14

Repeating the study on tumor size and and survivability in Lug cancer

Question 15

Explain the image

Answer 15

HE staining = tumor vascularity. IFNb decreased tumor vascularity

IHC: expression of IFNb (using antibodies to produce a dark dye)

TUNEL: TDT binds to broken DNA caused by apoptosis > brown appearance

TDT adds a labeled dinucleotide to the exposed OH group

Question 16

Talk me through the pathway of intrinsic mitochondrial apoptosis

Answer 16

Apoptotic stimulus > activate BH3 > make channel in membrane of mitochondria > release of cytochrome C > create apoptosome > activate exacutionaer caspases > apoptosis