The Effects of Reverse-Transcriptase Inhibitor Analogs on ECM Synthesis and Fibroblast Cells

1. The Effects of Reverse-Transcriptase Inhibitor Analogs on ECM Synthesis and Fibroblast Cells Michael Bushnell Pittsburgh Central Catholic High School 11th Grade

2. Background Narrative • Patients with HIV/AIDS around the world are prescribed medications called RTIs (reverse-transcriptase inhibitors) • There are many different RTI options doctors can choose to prescribe to their patients given certain factors (severity of symptoms, time since diagnosis, strength of the patient, etc.) • HIV can be transmitted through contact between an open wound and infected matter • In certain areas around the world (i.e. developing nations), this type of transmission is common • How do we solve this problem?

3. Experimental Purpose • “To kill two birds with one stone” • If certain RTIs are more effective at tissue regeneration than others, it would only make sense to consider prescribing these RTIs to patients with HIV in transmission prone areas • In this way, the RTI would • 1) serve its purpose to treat HIV/AIDS • AND 2) assist in wound healing to prevent further transmission

4. Tissue Engineering What is Tissue Engineering? • The development of artificial implants, laboratory grown tissues, and genetically engineered cells and/or molecules to simulate the function of defective or injured tissues Why is Tissue Engineering significant? • Has the ability to simulate how tissues adapt and react to external and internal stimuli Applications: • Replacement or supplementation of tissue • Trauma, Damage, and Aging • Solutions to hereditary/congenital defects and conditions

5. Fibroblast Cells (3T3 Line) • a type of cell that synthesizes the extracellular matrix (ECM) and collagen; makes up the stroma • ECM: molecules secreted by cells that provides structural and biochemical support to the surrounding tissue • plays a critical role in wound healing • most common connective tissue in mammals 3T3 Cell Line • Has become the standard fibroblast cell model • This means they model the fibroblast cells in mammals and can be used to simulate effects in human cells

6. Reverse-Transcriptase Inhibitors (RTIs) • class of antiretroviral drugs used to treat HIV/AIDS • RTIs inhibit activity of reverse transcriptase, a viral DNA polymerase that is required for replication of the HIV virus in the process of transcription Mechanism of Action • Host cells phosphorylate RTIs to their respective nucleotide analogs • Analogs act as chain terminators of viral and host DNA, causing the desired antiviral effect and toxicity side effects

7. Selection of Experimental Variables • In this experiment, the nucleotide analogs are employed as the experimental variables • There are 4 nucleotide analogs • Thymidine • Adenosine • Guanosine • Cytidine • Because there are virtually two major mechanisms of action, one RTI from each group was chosen (based on availability, usage, and cost) Similar mechanism of action Similar mechanism of action

8. Significance of Application • Each variable group represents a drug prescribed to patients in the status quo • 0.1x Concentration Guanosine = entecavir (≈1 mg/mL dosage) • 1x Concentration Guanosine = abcavir (300 mg/mL dosage) • 0.1x Concentration Thymidine = stavudine (≈1 mg/mL dosage) • 1x Concentration Thymidine = zidovudine (300mg dosage) • It follows that the effects of the presence/combination of the above variables on the model will translate to the RTI’s effect in human fibroblasts

9. Primary Question Do RTI analogs exhibit a significant effect on fibroblast synthesis, and if so, to what extent do thymidine RTI analogs and/or guanosine RTI analogs at varying concentrations exhibit a beneficial or detrimental effect?

10. Hypothesis Null: The adenosine and thymidine RTI analogs will not work independently or synergistically to cause a change in the proliferation of the 3T3 cells and the synthesis of ECM outside of chance. Alternative: (Single Formal Hypothesis) The guanosine and thymidine RTI analogs will work independently and/or synergistically to cause a change in the proliferation of 3T3 cells and the synthesis of ECM outside of chance because of the toxic effects of reverse-transcriptase inhibition.

11. Materials List • Cryotank • 75mm^2 tissue culture treated flasks • 25 mm^2 tissue culture treated flasks • 250 mg guanosine dissodium salt hydrate • 250 mg thymidine dissodium salt hydrate • Spectrophotometer (595 nm) • Fetal bovine serum (FBS) • 3T3 Fibroblastic Cell Line (quantity in procedure) • Trypsin-EDTA • Pen and tape • Macropipette + sterile macropipette tips (1 mL, 5 mL, 10, mL, 20 mL) • Micropipettes + sterile tips • DMEM Serum - 1% and Complete Media (4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium • pyruvate, and 1500 mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete]) • 75 mL culture flask • Incubator • Nikon Inverted Microscope with imaging technology • Laminar Flow Hood • Laminar Flow Hood UV Sterilizing Lamp • Sharpie pen • Hemocytometer • Sterile PBS • Deoxycholate 4% • Ethanol (70%) • Sterile Filter • Cell scraper • Sterile Water • Nitrile gloves

12. Experimental Procedure Cell Preparation • A 1 mL sample of 3T3 cells (stored in Cryotank) was used to introduce 30 mL of 10% serum DMEM media in a 75mm^2 culture flask • The media was replaced with 15mL fresh media to remove fluid and was incubated (37° C, 5% CO2) for 2 days. Cell Density Range: 10^6 to 2x10^6 cells/mL • This culture was passed into 18 flasks in preparation for experiment and incubated for 2 days (37° C, 5% CO2) Variable Preparation • Two 10mL 100x stocks were made based on RTI dosages • 250mg of guanosine and thymidine salt dissolved in 10mL of media, respectively

13. Experimental Procedure (Proliferation) Proliferation Procedure • Seeded 18 flasks with 3T3 cells from the original flasks in 5mL of 10% DMEM media each • Allowed cells to incubate in CO2 incubator overnight so that they can adhere to the bottoms of the flasks • The next day each flask was: • Rinsed with 1mL of trypsin, pipetted out • Added 1mL trypsin, incubate for 5 minutes • Slap flask and confirm with microscope that cells are no longer adhered to bottom of flask • Quenched reaction with 5mL media. • Added variable as follows

14. Preparing the Variable Concentrations (Grid of Nine Model) 1 Control Flask + 8 Variable Flasks = 9 flasks x2 (for days 1 and 2)

15. Experimental Procedure Cell Counts • Half of the flasks were removed from the incubator (These are the day 1 flasks, one for each of the 9 groups) • Cell Counts Taken: • Hemocytometer loaded with 25 microliters on each grid (every hemo can be used for two replicates) • Repeated 4 time • 8 total replicatesfrom each flask, 144 total • Counted cells in field of view of hemocytometer under Nikon inverted microscope and multiplied count by 10^3 for total cells/mL 3. Repeated on Day 2 with the other 9 flasks

16. Experimental Procedure ECM Synthesis 1. Rinsed cells (18 total wells) with 1 mL PBS, removed it 2. Added deoxycholate, rocked at room temperature for 20 minutes 3. Removed deoxycholate, added 1 mL PBS 4. Used cell scraper to pool ECM into tube 5. Prepared 3-5 dilutions of a protein standard. 6. Pipetted 100 μL of each standard into test tube. 7. Added 5mL of diluted dye reagent to each tube, vortex. 8. Incubated at room temperature for 5 minutes to increase absorbance. 9. Measured absorbance (595 wavelength).

17. Analytical Goals • Do the RTI analogs significantly affect the proliferation of the fibroblasts/synthesis of ECM? • Does it appear that there exists a synergistic effect between the guanosine and thymidine analogs? • Which RTI analog/combination of RTI analogs produces the least toxic effect on fibroblast proliferation?

18. Imaging No Guanosine 0.1x Guanosine 1x Guanosine No Thymidine 0.1x Thymidine 1x Thymidine

19. Proliferation Results (All Groups) y-axis (# of cells times 10^4) x-axis (groups)

20. Proliferation Results (RTI Individual Comparisons) y-axis (# of cells times 10^4) x-axis (groups)

21. Proliferation ANOVAs (Interaction) • Interaction p-value is <0.05 at both days, indicating significant interaction between the two RTIs • Descending p-values indicate that interaction increases with time

22. Proliferation ANOVAs (Individual) Guanosine Thymidine Day 1 Day 2 Each p-value is <0.05, so statistical analyses indicate that both RTI analogs individually have significant effects on 3T3 proliferation to some degree.

23. Statistical Analysis: Dunnett’s Test • M(ean) S(quared) derived from ANOVA • Td: t-value • If t-value is greater than f-crit value (derived from ANOVA), then that group exhibits a significant effect.

24. Dunnett’s Test Results f-crit = 3.4668

25. ECM Assay Results (All Groups) y-axis (absorbance) x-axis (groups)

26. ECM Quantification Absorbance: quantification of the ratio of incident to transmitted spectral radiant power through a material measured by a spectrophotometer

27. High ECM Growth Low ECM Growth

28. ECM Assay Results (RTI Individual Comparison) y-axis (absorbance) x-axis (groups)

29. ECM Assay ANOVA (Interaction) Interaction p-value <0.05 indicates that these two RTIs affect ECM synthesis synergistically.

30. ECM Assay ANOVAs (Individual) Thymidine Guanosine The p-value of the ANOVA analysis of Guanosine is <0.05, indicating that guanosine had a significant negative effect on the synthesis of ECM. The p-value of the ANOVA analysis of Thymidine is >0.05, indicating that thymidine did not show a significant effect on the synthesis of ECM.

31. Dunnett’s Tests Results F-crit = 3.68232

32. Summary of Statistical Conclusions Cell Proliferation • All RTI analogs at both concentrations have individually significant negative effects on 3T3 cell proliferation with the exception of the day 2 thymidine groups (Accept Alternative Hypothesis) • Guanosine toxicity increases with time whereas thymidine toxicity decreases • RTI analogs exhibit significant interaction which increases with time ECM Assay • Guanosine at a high concentration exhibits a significant toxic effect on the synthesis of ECM • Interaction between RTIs is significant • Higher concentrations have a greater negative impact on ECM synthesis than cell proliferation

33. Applicable Conclusions RTIs cause wounds to heal slowly and makes transmission more likely. Fibroblast growth should be a prescription factor. • Overall, RTIs negatively impact cell proliferation and ECM synthesis • Guanosine at a high concentration is consistently significant, increased toxicity with time • Thymidine consistently exhibits the least toxic effects, especially long-term • Significant Interaction Guanosine analog RTIs like entecavir and abcavir should not be prescribed to patients in transmission prone areas. Thymidine analog RTIs like stuvudine and zidovudine should be prescribed to patients in transmission prone areas. If a patient is or has taken two RTIs, fibroblast regeneration is negatively impacted.

34. Limitations and Extensions Limitations and Obstacles • Counting-Human Error • Cell Death • Costs • Equipment • Availability of Materials Extensions • Use the actual RTI medications (need research certification) • Clinical Trials with Fibroblastic Monitoring • Cytidine and Adenosine Analogs • Cost-Benefit Analysis • Automated Cell Counter • HIV Vaccine Testing

35. Works Cited • https://www.poz.com/drugs/classes/Nucleoside-Nucleotide-Reverse-Transcriptase-Inhibitors • https://www.nibib.nih.gov/science-education/science-topics/tissue-engineering-and-regenerative-medicine • http://www.sciencedirect.com/science/article/pii/S0169409X98000210 • https://www.technologyreview.com/s/600763/10-breakthrough-technologies-2016-immune-engineering/ • https://www.ncbi.nlm.nih.gov/pubmed/16640100 • https://www.drugs.com/dosage/abacavir.html • https://www.drugs.com/dosage/entecavir.html • https://www.drugs.com/dosage/stavudine.html • https://www.drugs.com/dosage/zidovudine.html