St. Josephs Cancer Research

1. St. Josephs Cancer Research The Search for the Cure

2. Acknowledgement Hill Park Secondary School, The Co-Op & Science Departments and Mr. Watts wish to thank Dr. D. Tang for allowing Brandon Tate to do a Co-Op placement in his lab (McMaster University/St. Joseph’s Hospital) and Andrew, a PhD student in Dr. Tang’s lab who both graciously gave of their time to supervise Brandon’s work on a daily basis.

3. What I Expected to Learn From This Placement From my placement I expected to learn many new lab protocols and generally immerse myself amongst professionals in the laboratory setting.

4. General Terms • Oncogene – a.k.a. the accelerator, they are genes that cause rapid cell division • Medium – a nutrient rich fluid that includes an antibiotic to ensure cell growth and specificity • PIN – prostatic intraepithelial neoplasia • Tumor Suppressor – a.k.a. the brakes, they are genes that slow cell division • Proliferation – rapid cell division • Antibody – a protein that is formed by the immune system that binds to foreign material

5. Facts About Cancer • Cancer is a mutation in DNA that allows cells to proliferate with no bounds • Approximately 92% of prostate cancer occurs in men over the age of 55 • EVERY man will have some degree of prostate cancer but not all will be life threatening • A tumor may grow without any nutrition and minimal air • A tumor secretes a certain substance that creates its own blood vessel network which attaches to the persons circulatory system

6. Subcloning How it works • A plasmid (circular DNA) is cut at specific sites by restriction enzymes • It is then linear and accepts other genes easier • The gene to be transferred is cut out of another plasmid by restriction enzymes which are hopefully similar to the ones used to cut the other plasmid • If they are not the same restriction enzymes, alternate steps must be performed to insert the target gene into the plasmid • We then confirm the cuts by running a DNA gel • Through many more processes we purify the newly formed plasmid and introduce it into a cell • An antibiotic resistant gene is also included in the cell to guarantee that only cells with our plasmid are the ones we are studying

7. Subcloning Use of subcloning • Subcloning is used to control the expression of a gene Some websites for illustrated learning of subcloning • http://www.biology.ualberta.ca/facilities/multimedia/uploads/microbiology/ecoli.swf • http://www.101science.com/Video.htm

8. Sample Procedure 1: 50L Reactions 2: 6L Sodium Acetate 3: 120L 100% Ethanol 4: 176L in -20 for 5 min 5: Spin +4-13000 RPM for 10 min 6: Remove supernatant 7: Wash 500L 70 % Ethanol 8: Spin +4-13000 RPM for 10 min 9: Remove Supernatant 10: Vacuum centrifuge to dry 11: 43L H2O to solve precipitate DNA (room temp. 5 min) 12: 5L pfu buffer 13: 10L DNTP 14: 1L pfu 15: 50L => PCR machine for 30 min 16: add 50L H2O 17: 100L chloroform 18: mix 200L hand 1 min 19: mix top speed 1min in centrifuge 20: keep supernatant 21: repeat steps 5-10 22: 43L H2O 23: 5L BAMH1, buffer III 24: 2L BAMH1 25: 50L +37 incubator overnight

9. Immunohistochemistry Immunohistochemistry is a process that involves visible dyes and antibodies

10. Immunofluorescence • Immunofluorescence is a process that involves invisible dyes and antibodies • The light emitted by the tissue may only be seen under different wavelengths of fluorescent light • At this lab we currently have three different stains which fluoresce blue, green, and red when viewed under the microscope • The blue stain is typically used for marking the nucleus • The other two differ between experiments • Tissue samples actually “auto-fluoresce” which generally means that they emit their own fluorescent light without actually being stained • This “auto-fluorescence” is deemed background and is a formidable opponent for any researcher • The goal of staining slides with immunofluorescent dyes is to target enzymes at specific sites in cells • In order to achieve this we must obtain signal over background

11. Auto-fluorescence Chart

12. Immunofluorescence Protocol • Immunohistochemistry Protocol- (Part 1) • Deparaffinize and rehydrate • 3 x 10min in xylene; 2 x 2min in 100% EtOH, 2 x 2min in 70% EtOH. • Rinse slides gently with ddH20 squirt bottle (be careful not to rinse directly on section). Wash with ddH20 in Coplin jar for 2 x 2min on shaker at 50rpm.

13. Quenching Endogenous Peroxidase • Wipe off excess H2O • Cover section with 3% H2O2 for 10min (This step is to essentially eliminate all of the peroxidase that is endogenous to the tissue thereby stopping non-specific binding.) • Wash with 1 x PBS in squirt bottle and then in coplin jar for 2 X 2 min on shaker at 50 rpm

14. Antigen retrieval • Pre-heat Antigen Retrieval Buffer in food steamer for at least 10-15min. • Incubate slides in pre-heated Antigen Retrieval Buffer for 20min. (this process breaks aldehyde crosslinks which were formed at the time of preservation by using heat and a citrate buffer) • Remove lid from food steamer and let slides sit for 10 min. • Remove entire slide box (with buffer) and let sit at room temperature on bench for 20min. • Wash slides with I x PBS for 3 x 5 min in Coplin jar on shaker.

15. Blocking and primary antibody addition • Gently dry slides and put in humidity chamber. • Blocking buffer: 3% normal goat serum, 3%BSA with I x PBS • Incubate slides for I hr at room temperature with blocking buffer: (blocks non-specific binding sites giving us a more accurate result) • Wipe and shake off excess blocking buffer. • Add primary antibody diluted in blocking buffer and incubate overnight at 4°C. (primary antibody binds to antigens on target)

16. Immunohistochemistry Protocol- (Part 2) • Wash slides 3 x 5 min PBS • Add biotinylated secondary antibody for 1 hr (secondary attaches to primary antibody) • Prepare ABC (Avidin-Biotin Complex) reagent let sit for 30 min • Wash slides 3 x 5 min PBS • Add ABC reagent, incubate for 1 hr (The avidin and biotinylated peroxidase provide an amplification of the signal ) • Wash slides 3 x 5 min PBS • Add DAB (Diaminobenzidine) solution, times range from 2-10 min (DAB forms a precipitate when reacted with peroxidase giving us our brown signal) • Wash slides with ddH2O for 30 sec and incubate in coplin jar for 5 min • Counterstain with haemotoxylin for as short as possible • Wash slides with ddH2O for 30 sec and incubate in coplin jar for 5 min • Clear and dehydrate slides (2 x 3 min 70% EtOH, 2 x 3 min 100% EtOH, 2 x 3 min xylenes • Coverslip with cytoseal mounting medium

17. Immunofluorescence Protocol- (Part 2) • Wash slides 3 x 5min in I x TBST • Add appropriate fluorescent secondary antibody (1:50) to each section. Small volume: (20L per I mL blocking buffer), Incubate in chamber for 1 hr at room temperature • Wash slides for 3 x 5min in l x TBST • Clear and dehydrate slides (opposite of depariffinize and rehydrate) • Coverslip with vectashield mounting medium (contains the DAPI which stains the nucleus blue) Immunofluorescence follows the same immunohistochemistry protocol. The only differences are immunofluorescence does not have a quenching endogenous peroxidase step and also instead of PBS we wash the slides with TBST, a detergent.

18. Conclusions In my extensive immunhistochemistry staining and analyzing of slides for CDK 11 and FADD we have learned that CDK 11 localizes to the cytoplasm in prostate carcinoma as compared to a more nuclear localization in a normal prostate gland. FADD co-localizes with the CDK 11 pattern in prostate samples. This result suggests that the kinase, CDK 11, and the adapter FADD, work together in the prostate. In my analysis of BCL2 ectopically expressing MCF7 (BCL2 null), BCL2 correlated with P-ATM, P-P53, and P-CHK2. This indicates activation of a DNA damage / tumour surveillance pathway. Included in my conclusions is a chart which, at the moment, includes the % of PIN, Ca, and N glands on a slide.

20. Immunohistochemistry Pictures Thin, elongated basal cell layer Prominent luminal cell layer CDK11 - Normal

21. FADD - Normal

22. CDK11 - PIN Degradation of basal cell layer Cells proliferating into lumen

23. FADD - PIN

24. CDK11 - Carcinoma Non-existent basal cell layer Small glands Loss of contact inhibition

25. FADD - Carcinoma

26. Immunofluorescence Figures Bcl-2 P53 Merge DAP1 Merge + DAP1

27. Bcl-2 CHK2 Merge DAP1 Merge + DAP1

28. Bcl-2 ATM Merge DAP1 Merge + DAP1

29. What I Learned From my experiences in the lab I have learned an abundance of techniques and skills that without this placement I would not have learned for many years to come. Everything I learned in the lab (immunofluorescence, immunohistochemistry, subcloning, how to put together figures, all the abbreviations that are included in a research discussion, etc.) I will be able to use in the coming years of university. If I had a chance to change one thing it would be my organization, which constantly plagues my work.

30. Thank you Thank you to everyone in the lab for all of the help and dealing with my constant questions. A special thank you to Dr. Tang for allowing me this amazing opportunity to work alongside your gifted employees and for bringing me one step closer to my life goals.