5. Cell – Counting Mechanism
• Recent Studies
• Problem
Abnormally large
Amount of cells
Apoptosis initiated
to decrease
tissue size
Large-cell Phenotype:
Normal amount of cells
Apoptosis not initiated
to decrease tissue size
6. Big Biological Question
• Can abnormal tissue growth from the
large-cell phenotype be suppressed
by forcing these cells into mitosis?
• Could an increase the number of cells
possibly alert the cell-counting
mechanism?
Pathways and Genes of Interest: Rheb-gene, myc-gene, and PI3K
pathway.
7. Genes and Pathways of Interest
• Rheb/Myc-genes and the PI3K pathway
• Overexpression = increase in cell mass with
failure to promote entry into mitosis.
• CDC25 or stg (Drosophila melanogaster)
• Phosphatase that activates mitotic cyclin-
dependent kinases, which force cells into
mitosis.
9. = Cell Death (Apoptosis)
Normal Tissue
+Rheb,Myc, or PI3K
+Rheb,Myc, or PI3K +stg
Cell-Counting Mechanism
10. Protocol Performed
• UASGal4 driver system: localizes phenotypic effects to certain
region
• enGal4: posterior compartment of wing
• Four sets of crosses:
• Wing tissue analysis based on two characteristics:
• 1. Size of the posterior compartment
• 2. Cell density in each compartment.
• Caspase stain to potentially identify apoptosis.
Wild-type x enGal4
x enGal + stg
Rheb, myc, or PI3K x enGal4
x enGal + stg
16. Figure 4. The introduction of stg to cells overexpressing growth pathways
increased the cell density within the posterior compartment. Cell density within a
surface area of 40 μm2 in the posterior and anterior compartments. When stg is
expressed the cell density in the posterior compartment is very similar to the
anterior compartment, indicating an increase in mitotic activity.
0
10
20
30
40
50
60
70
80
enG4,WT enG4/stg,WT myc myc (+stg) Rheb Rheb (+stg) PI3K PI3K (+stg)
CellCount
Posterior
Anterior
17. Interpretation of Results
• Overexpression of gene or pathway caused an
increase in cell size and overall tissue size.
• Cells did not enter mitosis
• When stg was introduced to each cross, tissue
size decreased, but cell count increased.
• Forced entry in mitosis may have alerted cell-
counting mechanism.
18. Ongoing and Future/Summer
Work
• Continue antibody staining
• Negative controls showed no apoptosis.
• Do antibody staining for stg-crosses.
• Verify apoptosis is biological process for
decreased tissue size.
• Inhibit apoptosis to see growth patterns.
• Look at tumor development from a different
biological approach
• Mutate a few cells rather than entire region.
19. References
• Chen BJ, Wu YL, Tanaka Y, Zhang W (2014) Small Molecules Targeting
c-Myc Oncogene: Promising Anti-Cancer Therapeutics. International
Journal of Biological Sciences 10(10); 1084-1096.
• Gao Y, et al (2000). Interplay of p53 and DNA-repair protein XRCC4 in
tumorigenesis, genomic stability and development. Nature 404: 897-
900.
• Dang CV. (2012) Myc on the Path to Cancer. Cell Press 149 (1): 22-35.
• Cantley, L. (2002) The Phosphoinositide 3-Kinase Pathway. Science
296 (1655).
• Shaw, RJ., Cantley, LC. (2006) Ras, PI(3)K and mTOR signaling controls
tumour cell growth. Nature 441: 424-430.
• Nilsson, I., Hoffmann, I., (2000) Cell cycle regulation by the CDC25
phosphatase family. Prog Cell Cycle Res. 4: 107-114.
