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

Introduction and Goals

• 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.

Background• Genes are essential mechanisms for controlling cancer.

• Tumor hypoxia leads to resistance to cancer treatment since they are less well-oxygenated 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)

CDKs and Cyclins by Cell Cycle Phase

Science Watch

• 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.

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).

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)

Results

• Analyzed results

• 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.

Data: Ink4a and Arf24 hours Ink4a-1

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P7NP P7NPH10

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5 p=0.055

24 hours Ink4a-2

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P7NP P7NPH10

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48 hours Ink4a-1

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P7NP P7NPH10

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48 hours Ink4a-2

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P7NP P7NPH10

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24 hours Arf-1

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P7NP P7NPH10

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48 hours Arf-1

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P7NP P7NPH10

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10 p=0.79

24 hours Arf-2

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P7NP P7NPH10

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48 hours Arf-2

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P7NP P7NPH10

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10 p=0.1572

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.

Data (continued)

• Other CDKs and cyclins tested: cdk2, cdk4, cdk6, ccna, ccnb, ccnd, ccne

• Did not have significant fold change (such as ccnb shown here)

24h Ccnb1-1

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P7NP P7NPH10

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48h Ccnb1-1

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P7NP P7NPH1-2

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8 p=0.8855

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.

• Overall, samples with hypoxia gene had less gene expression change, whereas sample without the gene had more gene expression change.

Conclusions

• 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.

Future Research

• 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

Citations

• 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.

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.