Grace Guo examined how the gene hypoxia-inducible factor-1α (Hif-1α) regulates mRNA expression of cell cycle regulators in mouse sarcoma cells exposed to ionizing radiation. Samples with and without Hif-1α were irradiated and mRNA expression over time was analyzed. The tumor suppressor genes Ink4a and Arf showed significant increased mRNA expression in samples with Hif-1α compared to those without. Most other cell cycle genes like Ccnb1-1 did not exhibit significant changes. The results suggest Hif-1α plays a role in responses to radiation by altering expression of key regulators.

1. Role of Hif-1α in mRNA Expression:
Profile of Cell Cycle Regulators in Mouse
Sarcoma Cells after Ionizing Radiation
Grace Guo
North Carolina School of Science and Mathematics
Durham, NC

2. Introduction and Goals
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Examine radiation’s effects on cancerous soft tissue sarcoma
Investigate how hypoxia-inducible factor-1α (Hif-1α) regulates mRNA expression of
cell cycle regulators that include tumor promoters and suppressors
Use tumor samples with and without Hif-1α gene to research how the gene plays a
role in mRNA expression in various cell cycle regulators
Unknown if to be many changes in cell cycle regulatory genes are expected in these
two sets of tumor cells after irradiation, but would be surprising to see no changes
or drastic changes in the samples without Hif-1α.

3. Background
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Genes are essential mechanisms for controlling cancer.
Tumor hypoxia leads to resistance to cancer treatment since they are less welloxygenated than normal cells.
Hypoxia-inducible factor-1α (Hif-1α) is involved in the induction of oxygen
regulated genes. Is a heterodimeric transcription factor consisting of an alpha (α)
and a beta (β) subunit (Goda, 2003).
Cyclin-dependent kinase inhibitor (CDKN) genes are a family of regulators that
control cell cycle progression through proteins that function at specific phases of
the cell cycle (Nowsheen, 2012)
Complex damage response pathway: regulates known responses such as cell-cycle
arrest and apoptosis (Zhou, 2000)

4. CDKs and Cyclins by Cell Cycle Phase
• Cyclin-dependent kinases (CDKs) and cyclins
(ccn) were used
• These proteins use different mechanisms to
control cell cycle, but are similar and not
specific for a particular phase of the cycle
• For comparison’s sake, CDK’s cyclins, such as
ccna1-1 and ccnb1-1, were also used as
specific primers to analyze gene expression.
Science Watch

5. Significance
• Understand the intersection between cell DNA damage response and repair
pathways
• Help advances in radiation therapy and cancer treatment
• Build to existing knowledge of functions of genes that regulate apoptosis
and other cellular death processes
• Minimizing radiation related toxicities is a priority if healthcare providers
want improved clinical outcomes of cancer treatment. (Baskar, 2011).

6. Methods
• Designed appropriate primers for genes to be tested for
• One set of tumor samples had the gene Hif-1α (P7NP). Another set did not
(P7NPH1).
• Both irradiated. RNA samples were collected at 00 hours, 02 hours, 24
hours, and 48 hours
• Used RNA/Reverse Transcriptase and cDNA protocols  complement DNA
• Performed rtPCRs (real-time Polymerase Chain Reaction)

7. Results
• Analyzed results
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Down-regulation? Up-regulation?
Fold change (measure of level of gene expression) in rtPCR relative to non-irradiated
control over time
Used α= 0.05 significance level
• All Arf and Ink4a genes were up-regulators and had notable fold change.
Arf-1 had more change than Arf-2
• Many cell cycle regulatory factors did not have changes in the mRNA level.

8. R e la t iv e F o ld - C h a n g e
t o n o n - ir r a d ia t e d c o n t r o l
8
1
0
P7NP
P7NPH1
4 8 h o u r s In k 4 a - 1
p = 0 .0 0 1
6
4
2
0
P7NPH1
8
4
3
2
1
P7NP
P7NP
R e la t iv e F o ld - C h a n g e
p = 0 .0 1 5 0
0
p = 0 .0 3 5 7
6
4
2
0
P7NPH1
t o n o n - ir r a d ia t e d c o n t r o l
4
P7NPH1
10
3
2
1
4 8 h o u r s In k 4 a - 2
R e la t iv e F o ld - C h a n g e
p = 0 .2 0
0
P7NP
p = 0 .7 9
8
6
4
2
0
P7NP
P7NPH1
t o n o n - ir r a d ia t e d c o n t r o l
2 4 h o u r s In k 4 a - 2
t o n o n - ir r a d ia t e d c o n t r o l
2
t o n o n - ir r a d ia t e d c o n t r o l
2 4 h o u r s In k 4 a - 1
R e la t iv e F o ld - C h a n g e
3
5
t o n o n - ir r a d ia t e d c o n t r o l
4
R e la t iv e F o ld - C h a n g e
p = 0 .0 5 5
R e la t iv e F o ld - C h a n g e
P7NP
R e la t iv e F o ld - C h a n g e
5
t o n o n - ir r a d ia t e d c o n t r o l
R e la t iv e F o ld - C h a n g e
t o n o n - ir r a d ia t e d c o n t r o l
Data: Ink4a and Arf
2 4 h o u r s A r f-1
2 4 h o u r s A r f-2
5
p = 0 .0 0 8 6
4
3
2
1
P7NPH1
0
P7NP
4 8 h o u r s A r f-1
10
P7NPH1
4 8 h o u r s A r f-2
p = 0 .1 5 7 2
8
6
4
2
0
P7NP
Data Set 1: P7NP (Hif-1a presence) vs P7NPH1 (Hif-1a not present) relative fold
change at 24 and 48 hours after irradiation. Results were significant if p < 0.05.
P7NPH1

9. Data (continued)
R e la t iv e F o ld - C h a n g e
t o n o n - ir r a d ia t e d c o n t r o l
2 4 h C c n b 1 -1
5
p = 0 .4 2 8 6
4
• Other CDKs and cyclins tested: cdk2,
3
cdk4, cdk6, ccna, ccnb, ccnd, ccne
2
1
0
P7NP
P7NPH1
R e la t iv e F o ld - C h a n g e
t o n o n - ir r a d ia t e d c o n t r o l
4 8 h C c n b 1 -1
8
p = 0 .8 8 5 5
6
4
2
0
-2
P7NP
P7NPH1
• Did not have significant fold change
(such as ccnb shown here)

10. Conclusions
• Most genes did not have significant mRNA expression changes before and
after irradiation
• Ink4a and Arf had significant mRNA expression changes
• Suggests that sarcoma samples with Hif1-α had more fold change between
24 and 48 hours than samples without Hif1-α.
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Overall, samples with hypoxia gene had less gene expression change, whereas sample
without the gene had more gene expression change.

11. Conclusions
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Many genes did not have significant change, which was expected
Would have be surprising if there were drastic changes everywhere or if there no
changes at all, since P7NPH1 (lacking Hif1-α) tumors are sensitized to irradiation
Disadvantage of RNA studies: protein translation does not occur exactly the same
way or at the same speed across all proteins.
Even if there was no RNA difference it does not mean that there was no difference
in protein expression.

12. Future Research
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Examine specific cell cycle stages and the expression of mRNA during those stages
Why Ink4a and Arf increase expression and what role they play
Investigate cell cycle regulators at the RNA and protein levels (western blotting),
not just at the mRNA levels
Quantify percentage of expression in each cell cycle phase
Experiment with cell senescence, or permanent cell-cycle arrest, to understand
how cells react to stress and damage from outside sources

13. Citations
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Basker R, Lee KA, Yeo R, Yeoh, KW. 2012. Cancer and Radiation Therapy: Current Advances and
Future Directions. International Journal of Medical Sciences. [Internet]. [2012, cited 2013 Sept
05] 9(3):193-199.
Goda N, Dozier SJ, Johnson RS. HIF-1 in cell cycle regulation, apoptosis, and tumor progression.
Antioxidants & Redox Signaling [Internet]. [2003, cited 2014 Jan 24] 5(4): 467-73.
Nowsheen S, Yang ES. 2012. The Intersection between DNA Damage Response and Cell Death
Pathways. Experimental Oncology [Internet]. [2012, cited 2013 Sept 24] 34(3):243-254.
Zhou BB, Elledge SJ. 2000. The DNA damage response: putting checkpoints in perspective.
Nature. [Internet]. [2000, cited 2013 Oct 4] Vol 408: 433-439.

14. Acknowledgements
I would like to thank the following people for their help:
• The Kirsch lab, Duke University
• Mentors: Dr. David Kirsch, Minsi Zhang, Lixia Luo. Duke University
• Dr. Shoemaker, research coordinator. NC School of Science and Math.