Investigation of the cell-counting mechanism response to the “large-cell” phenotype in Drosophila melanogasterPhi Sigma Symposium Presentation Bryston Nham Advisor: Leslie Saucedo, Ph.DSpring 2015

Normal Development of a Cell (Conlon and Raff, 1999)•Cell Growth •Cell cycle progression •Terminal differentiation•Apoptosis

Normal Development of a Cell (Conlon and Raff, 1999)•Cell Growth •Cell cycle progression •Terminal differentiation•Apoptosis

Tumor Development

Cell – Counting Mechanism

•Recent Studies

•Problem

Abnormally large Amount of cells

Apoptosis initiated to decreasetissue size

Large-cell Phenotype:Normal amount of cells

Apoptosis not initiatedto decrease tissue size

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.

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.

+Rheb,myc,PI3K+Rheb,myc,PI3K +stg

Cell-Cycle Diagram

No entry in mitosis

= Cell Death (Apoptosis)

Normal Tissue

+Rheb,Myc, or PI3K+Rheb,Myc, or PI3K +stg

Cell-Counting Mechanism

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

Results: Control Crosses enGal4 Anterior

Posterior

enGal4 (+stg) Anterior Posterior

Results: Rheb-gene crosses enGal4,Rheb Anterior Posterior

enGal4,Rheb (+stg) Anterior Posterior

Results: PI3K crosses enGal4, PI3K Anterior

Posterior

enGal4,PI3K (+stg) Anterior Posterior

Results: Myc-crosses enGal4,myc Anterior

Posterior

enGal4,myc (+stg) Anterior Posterior

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.

enG4,WT enG4/stg,WT myc myc (+stg) Rheb Rheb (+stg) PI3K PI3K (+stg)0

10

20

30

40

50

60

70

80

Posterior Anterior

Cell

Coun

t

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.

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.

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.

Thank you for your time! Questions?