Limits...
HDM2 antagonist MI-219 (spiro-oxindole), but not Nutlin-3 (cis-imidazoline), regulates p53 through enhanced HDM2 autoubiquitination and degradation in human malignant B-cell lymphomas.

Sosin AM, Burger AM, Siddiqi A, Abrams J, Mohammad RM, Al-Katib AM - J Hematol Oncol (2012)

Bottom Line: MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3.Additionally, this mechanism appears to correspond to biological outcome.Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Barbara Ann Karmanos Cancer Institute (KCI), Detroit, MI 48201, USA.

ABSTRACT

Background: Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, thereby compromising p53 activity. Therefore, lymphoma is a suitable model for studying the therapeutic value of disrupting the HDM2-p53 interaction by small-molecule inhibitors (SMIs). HDM2 have been developed and are under various stages of preclinical and clinical investigation. Previously, we examined the anti-lymphoma activity of MI-319, the laboratory grade of a new class of HDM2 SMI, the spiro-oxindole, in follicular lymphoma. Since then, MI-219, the clinical grade has become readily available. This study further examines the preclinical effects and mechanisms of MI-219 in a panel of human lymphoma cell lines as well as a cohort of patient-derived B-lymphocytes for its potential clinical use.

Results: Preclinical assessment of MI-219 was evaluated by means of an in vitro and ex vivo approach and compared to Nutlin-3, the gold standard. Characterization of p53 activity and stability were assessed by quantitative PCR, Western blot, and immunoprecipitation. Biological outcome was measured using Trypan blue exclusion assay, Annexin V/PI, PARP and caspase-3 cleavage. Surprisingly, the overall biological effects of Nutlin-3 were more delayed (48 h) while MI-219 triggered an earlier response (12-24 h), predominantly in the form of apoptotic cell death. Using a cell free autoubiquitination assay, neither agent interfered with HDM2 E3 ligase function. MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3. MI-219, but not Nutlin-3, enhanced the autoubiquitination and degradation of HDM2.

Conclusions: Our data reveals unexpected differences between MI-219 and the well-studied Nutlin-3 in lymphoma cell lines and patient samples. We suggest a novel mechanism for MI-219 that alters the functional activity of HDM2 through enhanced autoubiquitination and degradation. Additionally, this mechanism appears to correspond to biological outcome. Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance.

Show MeSH

Related in: MedlinePlus

HDM2 SMIs enhance p53 stability at the posttranslational level. A) WSU-FSCCL cells were exposed to 50 μM cyclohexamide (CHX) to stop protein translation or 10 μM MG132 to halt proteasome activity over the course of 4 h. B) Cells were pre-treated with 10 μM of Nutlin-3 (B1) or 10 μM MI-219 (B2) for 24 h and then exposed to 50 μM CHX for up to 4 additional hours. Samples were removed at 0.25, 0.5, 1.0, 2.0 and 4 h to evaluate the stability of p53 protein. RD represents relative density to time 0 for CHX and MG132 blots in (A) and time at 24 h 10 μM pre-treatment for blots in section (B). Changes in relative protein densities are plotted from values obtained (and listed below blots) to show effects of HDM2 SMIs on sustaining p53 protein expression in the presence of added 50 μM CHX.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3473265&req=5

Figure 6: HDM2 SMIs enhance p53 stability at the posttranslational level. A) WSU-FSCCL cells were exposed to 50 μM cyclohexamide (CHX) to stop protein translation or 10 μM MG132 to halt proteasome activity over the course of 4 h. B) Cells were pre-treated with 10 μM of Nutlin-3 (B1) or 10 μM MI-219 (B2) for 24 h and then exposed to 50 μM CHX for up to 4 additional hours. Samples were removed at 0.25, 0.5, 1.0, 2.0 and 4 h to evaluate the stability of p53 protein. RD represents relative density to time 0 for CHX and MG132 blots in (A) and time at 24 h 10 μM pre-treatment for blots in section (B). Changes in relative protein densities are plotted from values obtained (and listed below blots) to show effects of HDM2 SMIs on sustaining p53 protein expression in the presence of added 50 μM CHX.

Mentions: HDM2 inhibition is hypothesized to increase p53 stability by reducing HDM2-mediated degradation. However, p53 stability could also be the result of enhanced p53 protein translation. To demonstrate that upregulation of p53 protein expression shown in the Western blots were the result of HDM2 inhibition by SMIs, the half-life of p53 was monitored. The inhibition of protein synthesis by treatment with 50 μM CHX alone led to a marked decrease of p53 protein expression over time. Blocking protein translation with CHX decreased the turnover of endogenous p53 (Time 0-2 h; ~t1/2 = 0.68 h) (Figure 6). Furthermore, addition of 10 μM of the proteasome inhibitor MG132 alone ameliorates the degradation of p53, thereby enhancing its stabilization.


HDM2 antagonist MI-219 (spiro-oxindole), but not Nutlin-3 (cis-imidazoline), regulates p53 through enhanced HDM2 autoubiquitination and degradation in human malignant B-cell lymphomas.

Sosin AM, Burger AM, Siddiqi A, Abrams J, Mohammad RM, Al-Katib AM - J Hematol Oncol (2012)

HDM2 SMIs enhance p53 stability at the posttranslational level. A) WSU-FSCCL cells were exposed to 50 μM cyclohexamide (CHX) to stop protein translation or 10 μM MG132 to halt proteasome activity over the course of 4 h. B) Cells were pre-treated with 10 μM of Nutlin-3 (B1) or 10 μM MI-219 (B2) for 24 h and then exposed to 50 μM CHX for up to 4 additional hours. Samples were removed at 0.25, 0.5, 1.0, 2.0 and 4 h to evaluate the stability of p53 protein. RD represents relative density to time 0 for CHX and MG132 blots in (A) and time at 24 h 10 μM pre-treatment for blots in section (B). Changes in relative protein densities are plotted from values obtained (and listed below blots) to show effects of HDM2 SMIs on sustaining p53 protein expression in the presence of added 50 μM CHX.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473265&req=5

Figure 6: HDM2 SMIs enhance p53 stability at the posttranslational level. A) WSU-FSCCL cells were exposed to 50 μM cyclohexamide (CHX) to stop protein translation or 10 μM MG132 to halt proteasome activity over the course of 4 h. B) Cells were pre-treated with 10 μM of Nutlin-3 (B1) or 10 μM MI-219 (B2) for 24 h and then exposed to 50 μM CHX for up to 4 additional hours. Samples were removed at 0.25, 0.5, 1.0, 2.0 and 4 h to evaluate the stability of p53 protein. RD represents relative density to time 0 for CHX and MG132 blots in (A) and time at 24 h 10 μM pre-treatment for blots in section (B). Changes in relative protein densities are plotted from values obtained (and listed below blots) to show effects of HDM2 SMIs on sustaining p53 protein expression in the presence of added 50 μM CHX.
Mentions: HDM2 inhibition is hypothesized to increase p53 stability by reducing HDM2-mediated degradation. However, p53 stability could also be the result of enhanced p53 protein translation. To demonstrate that upregulation of p53 protein expression shown in the Western blots were the result of HDM2 inhibition by SMIs, the half-life of p53 was monitored. The inhibition of protein synthesis by treatment with 50 μM CHX alone led to a marked decrease of p53 protein expression over time. Blocking protein translation with CHX decreased the turnover of endogenous p53 (Time 0-2 h; ~t1/2 = 0.68 h) (Figure 6). Furthermore, addition of 10 μM of the proteasome inhibitor MG132 alone ameliorates the degradation of p53, thereby enhancing its stabilization.

Bottom Line: MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3.Additionally, this mechanism appears to correspond to biological outcome.Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Barbara Ann Karmanos Cancer Institute (KCI), Detroit, MI 48201, USA.

ABSTRACT

Background: Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, thereby compromising p53 activity. Therefore, lymphoma is a suitable model for studying the therapeutic value of disrupting the HDM2-p53 interaction by small-molecule inhibitors (SMIs). HDM2 have been developed and are under various stages of preclinical and clinical investigation. Previously, we examined the anti-lymphoma activity of MI-319, the laboratory grade of a new class of HDM2 SMI, the spiro-oxindole, in follicular lymphoma. Since then, MI-219, the clinical grade has become readily available. This study further examines the preclinical effects and mechanisms of MI-219 in a panel of human lymphoma cell lines as well as a cohort of patient-derived B-lymphocytes for its potential clinical use.

Results: Preclinical assessment of MI-219 was evaluated by means of an in vitro and ex vivo approach and compared to Nutlin-3, the gold standard. Characterization of p53 activity and stability were assessed by quantitative PCR, Western blot, and immunoprecipitation. Biological outcome was measured using Trypan blue exclusion assay, Annexin V/PI, PARP and caspase-3 cleavage. Surprisingly, the overall biological effects of Nutlin-3 were more delayed (48 h) while MI-219 triggered an earlier response (12-24 h), predominantly in the form of apoptotic cell death. Using a cell free autoubiquitination assay, neither agent interfered with HDM2 E3 ligase function. MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3. MI-219, but not Nutlin-3, enhanced the autoubiquitination and degradation of HDM2.

Conclusions: Our data reveals unexpected differences between MI-219 and the well-studied Nutlin-3 in lymphoma cell lines and patient samples. We suggest a novel mechanism for MI-219 that alters the functional activity of HDM2 through enhanced autoubiquitination and degradation. Additionally, this mechanism appears to correspond to biological outcome. Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance.

Show MeSH
Related in: MedlinePlus