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Role of the p21 Protein in the Fanconi Anemia (FA) Pathway Mae Shen Faculty Sponsor: Dr. Niall Howlett Fanconi Anemia - Clinical Description Congenital malformations – – – – – Short stature Microcephaly (small head) Micropthalmia (small eyes) Hypo/hyperpigmentation Abnormal thumbs Hematological abnormalities – Bone marrow failure – Acute myeloid leukemia (x800) Non-Hematological abnormalities – Head and neck squamous cell carcinomas (x2000) – Gynecologic squamous cell carcinomas (x4000) – Benign and malignant liver, brain and renal tumors Fanconi Anemia - Inheritance • Recessive genetic disorder – Biallelic mutations in any one of thirteen genes results in FA – Autosomal and X-linked • Incidence: 1 in 200,000 – 400,000 live births – Higher incidence rate in certain populations such as Spanish Gypsies and Ashkenazi Jews. Why Study Fanconi Anemia? • Link to Cancer – FA is strongly linked to cancer in the general (non-FA) population. – Four FA genes are breast cancer susceptibility genes in the heterozygous state, for example FANCD1/BRCA2. • Target for Cancer Chemotherapy – The FA pathway is a major cellular determinant of resistance to DNA crosslinks. – Oxaliplatin and cisplatin are DNA-crosslinking agents commonly used in cancer chemotherapy. – Drugs that inactivate the pathway could be used in adjuvant cancer chemotherapy. The Fanconi Anemia Pathway G F C A E 24 M L 100 Ub UBE2T Ub Ub D2 ATM B BLM I ATR ? D2 Ub USP1 Ub UAF1 ATRIP P Ub PCNA P Ub I NBS1 BRCA1 J/BRIP1 D1/BRCA2 51 N/PALB2 51 51 p21 is Required for DNA damage-inducible FANCD2 monoubiquitination HCT116 p21+/+ Hours post UV (20 J/m2) FANCD2-Ub FANCD2 0 0.1 0.3 0.6 1 p21 p21 -/2 6 24 * 0 0.1 0.3 0.6 1 2 6 24 * 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 What is p21? • Major regulator of the cell cycle • Arrests cell cycle 1) Inhibit CDKs 2) Interact with PCNA to halt DNA replication • Major target of the tumor suppressor transcription factor p53 Figure 8.4 The Biology of Cancer (© Garland Science 2007) My Project Goal: Characterize the interaction between FANCD2/FANCD2-Ub and p21 Specifically: 1) Are FANCD2 monoubiquitination and p21 coordinately regulated following DNA damage? 2) Is p21 required for resistance to DNA crosslinks? 3) Does inhibition of p21 degradation affect FANCD2 monoubiquitination? 4) Is the CDK-inhibiting function of p21 required for FANCD2 monoubiquitination? 1) Are D2 mono-Ub and p21 Coordinately Regulated following DNA Damage? Rationale: – If the kinetics of D2 mono-Ub and p21 expression are similar, this would be suggestive that both proteins are involved in the same cellular process. Methods: – Treat cells with DNA damaging agents. • UV irradiation (TT dimer) • Mitomycin C (DNA crosslink) – Observe levels of monoubiquitinated D2 (D2-Ub) and p21 over time. 1) Are D2 mono-Ub and p21 coordinately regulated following DNA damage? U2OS Hours post-UV (20J/m2 ) 0 0.3 1 2 6 24 1 2 3 4 5 6 0 0.3 1 2 6 24 1 2 3 4 5 6 FANCD2-Ub FANCD2 * p21 U2OS Hours post-MMC (100nM) FANCD2-Ub FANCD2 p21 * FANCD2 and p21 do not appear to be coordinated following DNA damage 2) Is p21 required for resistance to DNA crosslinks? Rationale: • FA patient cells are hypersensitive to DNA-crosslinks. • Since cells lacking p21 have impaired monoubiquitination of D2, they should exhibit a similar sensitivity. Methods: • Treat HCT116 p21+/+ and HCT116 p21-/- cells with the DNAcrosslinking agent mitomycin C (MMC). • Allow cells to grow for about 10 days. • Stain with crystal violet to quantify cells that survived. • Observe whether cells lacking p21 (HCT116 p21-/-) are more sensitive to MMC than wild type cells (HCT116 p21+/+). 2) Is p21 required for resistance to DNA crosslinks? 100 p21+/+ p21 -/- % Viability 80 60 40 20 0 0 100 200 300 400 MMC (nM) p21 does not appear to be required for resistance to MMC 3) Does inhibition of p21 degradation affect FANCD2 monoubiquitination? • Rationale: – p21 is degraded by the proteasome following UV irradiation. – If p21 degradation is inhibited, how is D2 mono-Ub affected? • Methods: – Treat cells with the proteasome inhibitor MG-132 prior to UV irradiation to inhibit p21 degradation. – Observe levels of FANCD2 monoubiquitination. 3) Does inhibition of p21 degradation affect FANCD2 monoubiquitination? Control Hours post-UV (20J/m2 ) No UV 2 MG-132 (10μM) 6 2 6 BJ-Tert FANCD2-Ub FANCD2 * p21 1 2 3 4 5 Hela FANCD2-Ub FANCD2 p21 U2OS FANCD2-Ub FANCD2 * p21 1 2 3 4 5 FANCD2 monoubiquitination decreases while p21 increases consistently after treatment with MG-132. 4) Is the CDK-inhibiting function of p21 required for FANCD2 monoubiquitination? Rationale: – One of the functions of p21 is to inhibit CDK. – Is this function required for FANCD2 mono-Ub? Methods: – Treat HCT116 p21-/- cells with roscovitine (a CDK inhibitor) prior to UV irradiation. – Observe whether roscovitine can substitute CDKinhibiting role of p21 and restore D2 monoubiquitination following DNA damage. 4) Is the CDK-inhibiting function of p21 required for FANCD2 monoubiquitination? Roscovitine (5 μM) Control Hours post-UV (20J/m2) 0 2 6 24 0 2 6 24 FANCD2-Ub FANCD2 HCT116 p21+/+ FANCD2-Ub FANCD2 HCT116 p21- / 1 2 3 4 5 6 7 8 1.4 Ratio Ub/Non-Ub 1.2 1 p21+/+ NT 0.8 p21+/+ Rosc 0.6 p21-/- NT 0.4 p21-/- Rosc 0.2 0 0 2 6 Hours Post-UV 24 Roscovitine does not restore UV-inducible FANCD2 monoubiquitination Conclusions p21 is clearly important to the FA pathway. However the interaction between FANCD2 and p21 is complex. The mechanism by which p21 regulates the monoubiquitination of FANCD2 remains to be uncovered. Thank You! RI-INBRE Leukemia Research Foundation Fanconi Anemia Research Fund Dr. Howlett Julie Jeanne Meg Fred Bill Sarah