Apoptosis/Cell Death, Cancer, Cell Cycle, Cell Division, Gene Regulation, Metastasis, Mitosis, Molecular Epidemiology, Nucleus, Oncogenes, Phosphorylation, Prostate, Protein Structure/Function, Transcription Factors, Transcriptional Activation and Repression, Viruses and Virology
Biophysics and Systems Pharmacology [BSP], Cancer Biology [CAB], Genetics and Genomic Sciences [GGS]
MS, Albert Einstein College of Medicine
PhD, Albert Einstein College of Medicine
Role of p53 in cell cycle checkpoints
p53 has clearly been implicated as a tumor suppressor in human cancer with mutation of the p53 gene being a common event. p53 has a well-characterized role in mediating the cellular response to DNA damage. The checkpoint in the G1 phase has been shown to be strictly p53-dependent. Due to the existence of a G2/M checkpoint that occurs in its absence, the precise role of p53 in preventing mitotic entry has been elusive. Ongoing studies are addressing this key aspect of p53 biology. The goal is to determine the molecular details of p53 function in this checkpoint in response to transient or sustained DNA damage. p53-dependent mechanisms of transcriptional regulation that affect mitotic entry and progression are being elucidated. The genes encoding mitotic regulators including Cdc25C, Survivin, Cdc20, and Cyclin B1 are targets for transcriptional downregulation. To elucidate the molecular basis, the regulation of the cdc25C gene is being studied as being representative. Insights gained in the detailed study of Cdc25C repression will then be examined for p53-dependent transcriptional downregulation of Survivin, Cdc20, and Cyclin B1. p53-dependent mechanisms of post-transcriptional regulation in response to DNA damage are also being studied. These include novel roles for Mdm2 in affecting the ability of p21CIP1 to inhibit cyclin-dependent kinases and in regulating Cdc25C protein stability. Preliminary data show that protein stability of not only Cdc25C, but also Survivin, Cdc20, and Cyclin B1 is regulated by p53. This intriguing finding is being further explored. The role of p53 and its downregulated targets in mitotic entry and progression after transient or sustained DNA damage is being examined. Upon transient treatment with DNA damaging agents wild-type p53 cells reversibly arrest and repair the damage, whereas p53 null cells fail to do so and die. The molecular basis for these effects will be elucidated. Ongoing studies address this central issue in understanding p53 function, namely how it mediates cell cycle checkpoints in response to DNA damage. The innovation of these experiments include the study of a new target for p53 that involves transcriptional repression occurring in a DNA binding-dependent manner, the novel observation that p53 regulates protein stability of particular mitotic regulators as part of checkpoint activation, and the intriguing possibility that Mdm2 plays a positive role as a downstream effector of p53-mediated cellular responses. The significance of this research relates to the clinical implications of selective targeting of tumor cells with a defective p53 pathway, especially given the frequency of p53 mutation in cancer. Taken together, these studies will elaborate a detailed understanding of how p53 mediates DNA damage checkpoints. This is expected to provide new avenues of pursuit that are relevant for prognosis and treatment of human cancer.
The C-terminal basic region of p53 in transcriptional regulation and tumor suppression
The goal is to achieve mechanistic understanding of the molecular interactions that regulate the tumor suppressor p53 as a master transcriptional regulator in response to genotoxic stress. Studies have shown that lysine acetylation of p53 plays an important role in the transcriptional activity of p53 that regulates cell cycle arrest, senescence or apoptosis. While multiple acetylation sites in p53 have been reported including K320, K373 and K382, as well as more recently, K120 and K164, specific effects of individual or combined acetylation of these lysine residues on p53 activity remain to be elucidated. It is hypothesized that distinct modifications of p53 including acetylation, methylation, ubiquitination and phosphorylation have differential regulatory effects on p53 transcriptional activity in conjunction with other cellular transcriptional proteins. These studies are expected to enhance our understanding of the molecular mechanisms underlying p53 functions in gene transcriptional regulation. Previous studies in vitro have led to conflicting hypotheses concerning the role of the C-terminal basic region of p53 in its ability to act as a transcription factor. In part, this confusion may be due to a reliance on ectopic overexpression approaches. Multiple approaches including a novel mouse model is being used to examine the requirement for the C-terminal basic region in p53-dependent outcomes in cells and in vivo. Approaches are being used to determine the molecular contributions of p300 and CBP in regulating p53-dependent transcriptional activity and cellular outcomes and to address the relevance of the interactions between p300 and CBP with the C-terminal basic region. The goal is to determine how specific co-activators and post-translational modifications contribute to p53-dependent gene expression as well as cellular outcomes and tumor suppression in vivo.
Transcriptional regulation of apoptosis
Resistance to apoptosis contributes to tumorigenesis as well as poor therapeutic outcome. The tumor suppressor p53 mediates two predominant cellular responses, cell cycle arrest and apoptosis, but the molecular basis for this cell fate determination has remained elusive. It is proposed that an interplay between p53-independent effectors and p53-dependent gene expression may be the crucial determinant for cellular outcome. To test this hypothesis the interplay between Sp/KLF family members and p53 on regulating gene expression is being examined, focusing on the role of p21CIP1 as an attenuator of the apoptotic response, and elucidating whether alterations in basal levels of expression of key members of apoptotic pathways contribute to cell fate decisions. Multiple p53 response elements located in both the promoter and first intron are involved in the DNA damage-induced upregulation of p21CIP1 with the two sites in the p21CIP1 promoter being regulated in distinct manners. The molecular basis for the differences between the two types of p53 sites is being elucidated and the interplay between p53, Sp1, and other Sp/KLF family members is being examined. The key role of other cellular factors such as Sp/KLF family members in p53-dependent gene regulation is being addressed as a means for influencing cellular outcomes. The ability of the cyclin-dependent kinase inhibitor p21CIP1 to interfere with apoptosis and influence cell fate has been suggested by published studies as well as preliminary data. Thus, the role of p21CIP1 in attenuating the cell death response is being validated and characterized and the underlying molecular basis for this intriguing effect of p21CIP1 is being elucidated. Regulating the basal levels of expression of key components of apoptotic pathways is an alternative mechanism for determining cell fate outcomes. Elements in the bax gene that confer constitutive transcriptional regulation of Bax have been identified. NHE1 is a novel p53 target gene that has been shown to regulate the anti-apoptotic effects of Bcl-XL via deamidation. The molecular interplay between Bax, NHE1, and Bcl-XL is being explored to determine whether such mechanisms can explain the apoptotic resistant phenotype of particular tumor cells. Depending upon particular cellular conditions, the tumor suppressor protein p53 induces growth arrest or mediates an apoptotic response. The optimal therapeutic response to DNA damage caused by many chemotherapeutic agents is cell death rather than inhibition of cell cycle progression. Elucidating the molecular mechanisms that are responsible for regulating the ability of p53 to trigger apoptosis versus arrest may lead to more effective therapeutic intervention and a way to overcome the chemotherapeutic-resistant phenotype found in many tumors.
St. Clair S, Giono L, Varmeh-Ziaie S, Resnick-Silverman L, Liu WJ, Padi A, Dastidar J, DaCosta A, Mattia M, Manfredi JJ. DNA damage-induced downregulation of Cdc25C is mediated by p53 via two independent mechanisms: one involves direct binding to the cdc25C promoter. Mol Cell 2004; 16: 725-736.
Manfredi JJ, Dong J, Liu WJ, Resnick-Silverman L, Qiao R, Chahinian P, Saric M, Gibbs AR, Phillips JI, Murray J, Axten CW, Nolan RP, Aaronson SA. Evidence against a role for SV40 in human mesothelioma. Cancer research 2005 Apr; 65(7).
Giono LE, Manfredi JJ. Mdm2 is required for inhibition of Cdk2 activity by p21, thereby contributing to p53-dependent cell cycle arrest. Molecular and cellular biology 2007 Jun; 27(11).
Varmeh-Ziaie S, Manfredi JJ. The dual specificity phosphatase Cdc25B, but not the closely related Cdc25C, is capable of inhibiting cellular proliferation in a manner dependent upon its catalytic activity. The Journal of biological chemistry 2007 Aug; 282(34).
Torti FM, Altieri D, Broach J, Fan H, Lotze M, Manfredi J, Matrisian L, Yu D, Giaccia A. Ph.D. Training in cancer biology. Cancer research 2008 Nov; 68(22).
Varmeh S, Manfredi JJ. Overexpression of the dual specificity phosphatase, Cdc25C, confers sensitivity on tumor cells to doxorubicin-induced cell death. Molecular cancer therapeutics 2008 Dec; 7(12).
Varmeh S, Manfredi JJ. Inappropriate activation of cyclin-dependent kinases by the phosphatase Cdc25b results in premature mitotic entry and triggers a p53-dependent checkpoint. The Journal of biological chemistry 2009 Apr; 284(14).
Manfredi JJ. The Mdm2-p53 relationship evolves: Mdm2 swings both ways as an oncogene and a tumor suppressor. Genes & development 2010 Aug 24(15).
Hamard PJ, Lukin DJ, Manfredi JJ. p53 basic C terminus regulates p53 functions through DNA binding modulation of subset of target genes. The Journal of biological chemistry 2012 Jun; 287(26).
Carvajal LA, Hamard PJ, Tonnessen C, Manfredi JJ. E2F7, a novel target, is up-regulated by p53 and mediates DNA damage-dependent transcriptional repression. Genes & development 2012 Jul; 26(14).
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