The study investigated how overexpression of the Rheb, myc, and INR genes in Drosophila melanogaster leads to increased cell size (the "large-cell" phenotype) and whether this activates the cell-counting mechanism. When stg was introduced alongside gene overexpression, it forced cells to divide, increasing cell counts and potentially alerting this mechanism. Results found stg increased cell numbers in posterior compartments with gene overexpression, decreasing cell size differences between compartments. This suggests stg activated the cell-counting mechanism, likely through initiating apoptosis to reduce tissue size, as supported by caspase-3 staining. The work provides insights into how oncogene amplification may evade apoptosis and how forced proliferation could counter this.

1. Investigation of the response to the “large-cell” phenotype caused by Rheb, myc, and
INR overexpression in Drosophila melanogaster
• Background Information
• Oncogenes have the potential to cause cancer when they are mutated or amplified (1).
Most normal cells undergo programmed cell death or apoptosis when certain functions are
altered, but activated oncogenes can cause cells designated for apoptosis to survive and
proliferate instead.
• Although some tumors develop due to rapid cell proliferation or an increase in the number
of cells, other tumors arise due to an increase in cell size.
• The amplification of certain genes (Rheb, myc, and INR) promote cell growth without
proliferation, creating a “large-cell” phenotype.
• Gal4 drivers are used to study phenotypic patterns caused by gene amplification in only
certain desired regions of an organism.
• The enGal4 driver creates phenotypic effects from amplification in the
posterior compartment of the fruit fly wing (2).
• Stg (parallel to CDC25 in humans) is a phosphatase that controls entry into and
progression through various stages of the cell cycle and is a rate-limiting factor for mitosis
(3).
• “Cell-counting” mechanism postulate: when there are deemed too many cells in a certain
region of the body, apoptosis will be initiated to eliminate the excess amount of cells.
Biological Questions
• If we replicate the “large-cell” phenotype to increase tissue size, can we alert the cell-
counting mechanism by forcing mitotic division with stg?
• Increase in cell count between stg being absent and present would indicate
forced cell division.
• If there is an increase in cell number, would apoptosis be activated in order to reduce
tissue size?
Figure 2. The introduction of stg to cells overexpressing growth pathways increased the cell number 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 number in the posterior compartment is very similar to the anterior
compartment, indicating an increase in mitotic activity.
Table 1. Introduction of stg promotes cell division which causes a decrease in cell size in the
posterior compartment relative to the anterior compartment. With stg absent, cells in posterior
compartment were on average 159.7% larger than cells in the anterior compartment. With stg present,
cells in the posterior compartment were on average 110% larger than anterior compartment cells.
Figure 3. Overexpression of
INR with stg present causes
cell number to increase due
to forced entry into mitosis.
Expressing INR alone caused
cells to be 155% larger in
posterior compartment. When
stg was introduced, cell size in
the posterior compartment was
only 115% larger than anterior
compartment. (A) Adult wing
with only INR overexpressed.
(B) 40 µm2 region of the
anterior compartment with
only INR overexpressed.
(C) 40 µm2 region of
posterior compartment with
only INR overexpressed. (C)
40 µm2 region of the anterior
compartment with only INR
overexpressed. (D) Adult wing
with INR overexpressed and
stg present. The posterior and
anterior compartments are
labeled. (E) 40 µm2 of the
anterior compartment with
INR + stg. (F) 40 µm2 of the
posterior compartment with
INR + stg.
Figure 1. Cell-counting
mechanism activated
when there are
an abnormal amount of
cells within a region of an
organism.
Each cell secretes a single
hair, allowing for
quantification of cell
quantity. Overexpression of
genes promote cell growth,
which does not alert the
cell-counting mechanism.
When stg is introduced into
lines with gene
overexpression, cells are
forced to divide, creating an
abnormal large amount of
cells, alerting the cell-
counting mechanism to
activate apoptosis and
eliminate excess cells.
Results and Discussion
• Offspring that had Rheb, myc, and INR overexpressed along with no stg had larger cells in
the posterior compared to the anterior compartment, creating a significant difference in the
number of cells between the two compartments.
• Indicated by the lower density of hairs in the posterior compartment.(Fig.
3)
• Offspring that had Rheb, myc, and INR overexpressed with stg present (+stg) had a more
relatively similar amount of cells between the posterior and anterior compartment.
• Larger cells created by gene overexpression were forced to divide by stg,
indicated by the increase in cell count in the posterior compartment and its
relative similarity to the cell count in the anterior compartment.
• Introduction of stg into Rheb-overexpressed offspring saw the largest decrease in cell size
while myc-overexpressed offspring saw the smallest relative decrease in cell size with the
introduction of stg.
• Wing discs from control crosses and Rheb,PI3K overexpression (-stg) did not have
apoptotic activity, indicating that cell-counting mechanism was not alerted.
Current Work
• Performing caspase-3 antibody staining to confirm that apoptosis is the biological process
responsible for decreasing tissue size when stg is present.
• Beginning clonal analysis of oncogene expression with and without stg.
• Instead of mutating an entire region of a wing, a few cells will be mutated
via heat shock in order to portray a more realistic mechanism of how
cancer arises.
Future Work
• Following confirmation that apoptosis is present via the antibody stain, two more sets of
crosses will be performed to see if apoptosis is responsible for tissue reduction.
• UAS DIAP and UAS p35: two genes that prevent apoptosis.
• If tissue regains its abnormal growth when apoptosis is inhibited then it
can be concluded that apoptosis is the biological process responsible for
tissue reduction and the cell-counting mechanism was alerted.
Literature Cited
1 Pierotti, M.A., Sozzi, G., Croce, C.M. (2003) Mechanisms of oncogenic activation. Holland-
Frei Cancer Medicine. 6th Edition.
2 Busson, D., Pret, A.M. (2007) Gal4/UAS targeted gene expression for studying Drosophila
Hedgehog signaling. Methods Mol Biol. (397) 161-201.
3 Shay, J.W., Wright, W.E. (2001) Cellular senescence as a tumor-protection mechanism: the
essential role of counting. Genetics and Development 11:98-103.
Acknowledgements
I would like to thank the University of Puget Sound and the University Enrichment Committee
for funding my research. I would also like to thank Leslie Saucedo and the other students in
her lab who have supported me through this project.
Method
1. Two control crosses were performed to identify if enGal4 or stg had any significant effect
on cell growth without gene amplification.
2. If enGal4 or stg alone had no significant effect on cell size, 6 experimental crosses were
executed with a gene amplified in each cross.
3. Wings of adult offspring were removed and mounted for analysis.
4. Regional comparison (posterior vs. anterior) of tissue size was performed.
5. Cell count was performed in each region by taking a set perimeter in each compartment and
counting the amount of hairs as each cell secretes a single hair.
6. Statistical analysis performed to identify differences in cell size when stg was absent or
present in the offspring.
7. Caspase-3 antibody staining performed to potentially identify apoptotic activity.
Male
Female
(Virgin)
Control
Cross
#1
Wild-­‐Type
x
enGal4
Control
Cross
#2
Wild-­‐Type
x
enGal4/stg
Variable Cross
Male (Gene/Pathway Amplified)
Female (Virgin)
1
UAS myc x
enGal4
2
UAS myc x
enGal4/stg
3
UAS Rheb x
enGal4
4
UAS Rheb x
enGal4/stg
5
UAS INR x
enGal4
6
UAS INR x
enGal4/stg
Phenotype
Posterior
Anterior
Percent
of
Posterior
Cell
Size
to
Anterior
Cell
Size
enGal4
49.4
51.1
103.4%
enGal4/stg
50.8
52.2
102.7%
myc
39.8
50.2
126.1%
myc
(+stg)
54.2
54.6
100.7%
Rheb
26.2
51.2
195.4%
Rheb
(+stg)
44.2
44
99.5%
INR
49.7
76.9
154.7%
INR
(+stg)
56.9
65.9
115.8%
Figure 4. Caspase-3 antibody stain
indicates cell-counting mechanism
and apoptosis were not initiated in
control crosses and when Rheb and
PI3K were amplified with stg absent.
Blue fluorescence (DAPI) represents
stained nuclei. Red flourescence (CY3)
represents potential caspase activity.
Lack of intense CY3- signal indicates
caspase was not present in significant
regions of the posterior compartment of
the imaginal wing disc. Offspring with
stg present in control cross created a
weak CY3 signal concluding that stg
without any oncogene amplification
does not cause enough cell division to
alert the cell-counting mechanism and
initiate apoptosis.
!
Normal Tissue and
Cells
Enlarged Tissue and Cells
Rheb, myc, or
INR
overexpression
Cell-Counting
Mechanism
NOT alerted
Rheb, myc, PI3K,
or INR
overexpression
(+stg)
Cell-Counting
Mechanism
alerted
0
10
20
30
40
50
60
70
80
90
enG4
(control)
enG4/stg
(control)
myc
myc
(+stg)
Rheb
Rheb
(+stg)
INR
INR
(+stg)
Cell
Count
Posterior
Anterior
A.
D.
B.
C.
E.
F.