An attractive therapeutic approach against cancers with wild-type (wt) p53 is the emergent class ... more An attractive therapeutic approach against cancers with wild-type (wt) p53 is the emergent class of small molecule MDM2 inhibitors, including the imidazoline nutlin-3 and more potent spiro-oxindole MI-63 (Ascenta Therapeutics). Both can rapidly increase intracellular p53wt levels and induce apoptosis or a senescence growth arrest, depending on the neoplastic status of the target cell. A critical question in the clinical development of such MDM2 inhibitors is their potential for normal tissue toxicity. In the absence of MDM2, p53 activation can cause fatal normal tissue pathology; and MDM2 inhibition can induce p53-dependent cell senescence in mouse fibroblasts. To assess the effects of MI-63 effects in human cells, we compared treatment (2.5-10 uM, 1-7 d) responses in p53wt malignant human mammary epithelial cells (MCF7) with those of p53wt normal human foreskin fibroblasts (HCA2). As expected, MI-63 had little effect on p53mut human cancer cells (MDA-231). However, against p53wt cancer cells and fibroblasts, MI-63 produced a dose-dependent increase in both nuclear p53 levels and reporter gene (p21) expression within 24 h. MCF7 cells responded with a dose- and time-dependent increase in apoptosis after 2 d exposure (vital dye uptake, cleaved PARP). By contrast, HCA2 cells responded with partially reversible growth arrest and a senescent-like phenotype (increased ROS, SOD2, and SA-βgal) within 3 days. A common early (24 h) MI-63 response by both the malignant and normal cells was nuclear loss of high-mobility group box-1 protein (HMGB1) associated with its active extracellular secretion. Acetylation of this damage-reporting cytokine, thought to be required for its secretion, was detected by western and mass spectrometry analyses. Although HMGB1 is generally pro-inflammatory and may be a harbinger of the senescence-associated secretory phenotype (SASP), MI-63 treated fibroblasts failed to exhibit induction of DNA damage foci or other features characteristic of SASP including IL-6 secretion. Surprisingly, MI-63 treatment of HCA2 beginning 24 or 48 h after a senescence-inducing dose of ionizing radiation (10 Gy) prevented enlarged cell morphology and produced a >80% reduction in SASP associated IL-6 secretion. These findings indicate that upregulation of p53wt in proliferating fibroblasts by MI-63, in the absence of DNA damage or other cell stresses, induces an incomplete senescence like growth arrest without a SASP response, consistent with our finding that p53 restrains SASP. In contrast, MI-63 upregulation of p53wt in malignant epithelial cells caused apoptotic cell death. The early release of HMGB1 from these dying cancer cells may modulate host innate immune response and/or recruit tumor infiltrating inflammatory cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4507. doi:10.1158/1538-7445.AM2011-4507
Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In addition to its well ... more Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In addition to its well established genome stabilizing and tumor suppressing role, p53 can also modulate cell metabolism and mitochondrial function. Small molecule MDM2 antagonists like nutlin-3 or MI-63 (Ascenta) are therapeutic prototypes that rapidly increase wildtype (wt) p53 levels in mammalian cells inducing apoptosis in cancer cells and growth arrest or senescence in non-malignant cells. We employed MI-63 (10 uM × 24h) to induce a near 10-fold increase in p53wt protein expression and compare its mitochondrial bioenergetic and metabolic consequences in malignant (MCF7) and non-malignant (MCF10A) human mammary epithelial cells, whose fates are differentially affected by MI-63. We utilized chromatin immunopreciptation-DNA sequencing (ChIP-seq; Illumina 3G sequencer) and expression microarrays (Affymetrix U133Av2) to identify genome-wide direct and indirect p53 target genes potentially affecting MCF7 mitochondrial structure and function. Within 24h of p53wt upregulation, 1903 genes were differentially expressed including 539 of the 2239 differentially bound direct p53 gene targets, leaving 1364 differentially expressed indirect p53 target genes. While p53 binding was not detected in the mitochondrial genome, GO analysis identified genes associated with the mitochondrial matrix (3 up, 28 down) and genes associated with the outer mitochondrial membrane (7 up, 10 down) as significantly enriched among the p53 regulated nuclear targets (EASE = 0.003, FDR p = 0.03 and EASE = 0.002, FDR p = 0.02 respectively). Real-time, kinetic measurement (Seahorse XF) of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) was performed in adherent cultures of MCF7 and MCF10A cells after comparable 24 h MI-63 induction of p53wt. In contrast to the apparent inhibitory effect of p53wt induction on MCF7 transcription of mitochondrial matrix genes, basal and spare mitochondrial respiratory capacity were stimulated ∼2-fold. This p53wt induced increase in ATP supply and OCR was caused by slightly higher OXPHOS and maximal recruitment of glycolysis, assessed by ECAR and simulated by cell supplementation with pyruvate. Unlike this metabolic and bioenergetic response in the malignant MCF7 cells, p53wt upregulation in the non-malignant MCF10A cells not only failed to stimulate OCR and ECAR but significantly depressed glycolysis. While it is generally thought that the metabolic function of p53wt is to favor energy production by mitochondrial OXPHOS and resist the shift to glycolysis that is characteristically seen in cancer cells, our findings suggest that malignant and non-malignant epithelial cells may have different metabolic and bioenergetic responses to upregulated p53wt. Further studies are needed to determine if these different p53wt responses contribute to the different cell fate responses induced by MDM2 antagonists like MI-63. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3797. doi:10.1158/1538-7445.AM2011-3797
Purpose: We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential... more Purpose: We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential biomarker and therapeutic target in breast cancer. eIF4E facilitates nuclear export and translation of specific, growth-stimulatory mRNAs and is frequently overexpressed in cancer.Experimental Design: Breast cancer cells were treated with ribavirin, an inhibitor of eIF4E, and effects on cell proliferation and on known mRNA targets of eIF4E were determined. eIF4E expression was assessed, at the mRNA and protein level, in breast cancer cell lines and in skin biopsies from patients with metastatic disease. Additionally, pooled microarray data from 621 adjuvant untreated, node-negative breast cancers were analyzed for eIF4E expression levels and correlation with distant metastasis–free survival (DMFS), overall and within each intrinsic breast cancer subtype.Results: At clinically relevant concentrations, ribavirin reduced cell proliferation and suppressed clonogenic potential, correlating w...
Proceedings of the 53rd ACM Technical Symposium on Computer Science Education V. 2, 2022
Past research has shown interactive animations, and those that use real-life analogies in particu... more Past research has shown interactive animations, and those that use real-life analogies in particular, can play an important role in providing the intuition required to understand Computer Science concepts. Nonetheless, the use of analogies continues to be under-explored in CS education compared to other STEM fields. To break the impasse, we aim to create and evaluate a set of interactive animations based on analogies to understand their efficacy. As our first addition, we have created an animation explaining the Buffer Overflow computer systems concept. Explaining the concept abstractly has had a track record of ineffectiveness in our department, since the concept of computer memory as a series of contiguous storage locations is so foreign to students. Instead, the animation uses the analogy of a dry-cleaning shop with a series of hangers to provide a concrete mental picture of computer memory. Students explore various dry-cleaning scenarios, in which customers drop off and pick up their laundry, to understand at their own pace when buffer overflows cause harm and when they are silently ignored. This animation: https://scratch.mit.edu/projects/571317697/ (and others) will be provided as an open educational resource to instructors to encourage the use of interactive analogies in their teaching, and to undergraduate and K-12 students.
Proceedings of the 53rd ACM Technical Symposium on Computer Science Education V. 2, 2022
It is a challenge to engage students when teaching them abstract and complex computer systems con... more It is a challenge to engage students when teaching them abstract and complex computer systems concepts, such as buffer overflow, memory management, concurrent execution, and process synchronization. Past research has shown that interactive animation and real-life analogies make STEM concepts more approachable and help students achieve better learning outcomes. Based on these findings, we introduce interactive analogies into learning the concept of buffer overflow. More specifically, we created a dry-cleaning shop animation tool (https://scratch.mit.edu/projects/571317697/) targeting K-12 and undergraduate students. To assess the effectiveness of our tool, we are in the process of conducting a user study, in which students use our animation tool to learn about buffer overflow and take pre- and post-assessment on the concept. Our goal is to make CS learning more accessible to diverse students, regardless of their background and age.
Cadmium is an environmental contaminant that enters the body through diet or cigarette smoke. It ... more Cadmium is an environmental contaminant that enters the body through diet or cigarette smoke. It affects multiple cellular processes, including cell proliferation, differentiation, and apoptosis. Recently, cadmium has been shown to function as an endocrine disruptor, to stimulate estrogen receptor α (ERα) activity and promote uterine and mammary gland growth in mice. Although cadmium exposure has been associated with the development of breast cancer, the mechanism of action of cadmium remains unclear. To address this deficit, we examined the effects of cadmium treatment on breast cancer cells. We found that ERα is required for both cadmium-induced cell growth and modulation of gene expression. We also determined that ERα translocates to the nucleus in response to cadmium exposure. Additionally, we provide evidence that cadmium potentiates the interaction between ERα and c-Jun and enhances recruitment of this transcription factor complex to the proximal promoters of cyclin D1 and c-m...
Purpose: We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential... more Purpose: We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential biomarker and therapeutic target in breast cancer. eIF4E facilitates nuclear export and translation of specific, growth-stimulatory mRNAs and is frequently overexpressed in cancer. Experimental Design: Breast cancer cells were treated with ribavirin, an inhibitor of eIF4E, and effects on cell proliferation and on known mRNA targets of eIF4E were determined. eIF4E expression was assessed, at the mRNA and protein level, in breast cancer cell lines and in skin biopsies from patients with metastatic disease. Additionally, pooled microarray data from 621 adjuvant untreated, node-negative breast cancers were analyzed for eIF4E expression levels and correlation with distant metastasis–free survival (DMFS), overall and within each intrinsic breast cancer subtype. Results: At clinically relevant concentrations, ribavirin reduced cell proliferation and suppressed clonogenic potential, correlating...
Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In addition to its well ... more Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In addition to its well established genome stabilizing and tumor suppressing role, p53 can also modulate cell metabolism and mitochondrial function. Small molecule MDM2 antagonists like nutlin-3 or MI-63 (Ascenta) are therapeutic prototypes that rapidly increase wildtype (wt) p53 levels in mammalian cells inducing apoptosis in cancer cells and growth arrest or senescence in non-malignant cells. We employed MI-63 (10 uM × 24h) to induce a near 10-fold increase in p53wt protein expression and compare its mitochondrial bioenergetic and metabolic consequences in malignant (MCF7) and non-malignant (MCF10A) human mammary epithelial cells, whose fates are differentially affected by MI-63. We utilized chromatin immunopreciptation-DNA sequencing (ChIP-seq; Illumina 3G sequencer) and expression microarrays (Affymetrix U133Av2) to identify genome-wide direct and indirect p53 target genes potentially affecting MCF7 mitochondrial structure and function. Within 24h of p53wt upregulation, 1903 genes were differentially expressed including 539 of the 2239 differentially bound direct p53 gene targets, leaving 1364 differentially expressed indirect p53 target genes. While p53 binding was not detected in the mitochondrial genome, GO analysis identified genes associated with the mitochondrial matrix (3 up, 28 down) and genes associated with the outer mitochondrial membrane (7 up, 10 down) as significantly enriched among the p53 regulated nuclear targets (EASE = 0.003, FDR p = 0.03 and EASE = 0.002, FDR p = 0.02 respectively). Real-time, kinetic measurement (Seahorse XF) of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) was performed in adherent cultures of MCF7 and MCF10A cells after comparable 24 h MI-63 induction of p53wt. In contrast to the apparent inhibitory effect of p53wt induction on MCF7 transcription of mitochondrial matrix genes, basal and spare mitochondrial respiratory capacity were stimulated ∼2-fold. This p53wt induced increase in ATP supply and OCR was caused by slightly higher OXPHOS and maximal recruitment of glycolysis, assessed by ECAR and simulated by cell supplementation with pyruvate. Unlike this metabolic and bioenergetic response in the malignant MCF7 cells, p53wt upregulation in the non-malignant MCF10A cells not only failed to stimulate OCR and ECAR but significantly depressed glycolysis. While it is generally thought that the metabolic function of p53wt is to favor energy production by mitochondrial OXPHOS and resist the shift to glycolysis that is characteristically seen in cancer cells, our findings suggest that malignant and non-malignant epithelial cells may have different metabolic and bioenergetic responses to upregulated p53wt. Further studies are needed to determine if these different p53wt responses contribute to the different cell fate responses induced by MDM2 antagonists like MI-63. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3797. doi:10.1158/1538-7445.AM2011-3797
Pathways downstream of PI3K/AKT/mTOR that regulate protein translation, ribosome biogenesis, tran... more Pathways downstream of PI3K/AKT/mTOR that regulate protein translation, ribosome biogenesis, transcription, metabolism and autophagy are essential to the formation and progression of many human cancers as evidenced by the clinical approval of such selective mTOR inhibitors as temsirolimus (CCI-779, Wyeth) and everolimus (RAD-001, Novartis). However, clinical limitations to the effective use of mTOR inhibitors include the lack of validated tumor marker(s) predictive of mTOR inhibitor responsiveness, mTOR inhibitor release of S6 kinase-mediated negative feedback resulting in secondary activation of PI3K/AKT, and transcriptional upregulation of the downstream effector eIF4E. Using breast cancer cell lines (MCF7, MCF7/HER2, BT474, SKBr3, MDA231) showing varying sensitivity to RAD-001 (IC50 values for MTT viability @ 5 d from <0.2 nM to >20 nM), we observe marked suppression of phos-p70S6K and secondary phos-AKT activation, with variable suppression of phosSer65-4E-BP1 and downstre...
An attractive therapeutic approach against cancers with wild-type (wt) p53 is the emergent class ... more An attractive therapeutic approach against cancers with wild-type (wt) p53 is the emergent class of small molecule MDM2 inhibitors, including the imidazoline nutlin-3 and more potent spiro-oxindole MI-63 (Ascenta Therapeutics). Both can rapidly increase intracellular p53wt levels and induce apoptosis or a senescence growth arrest, depending on the neoplastic status of the target cell. A critical question in the clinical development of such MDM2 inhibitors is their potential for normal tissue toxicity. In the absence of MDM2, p53 activation can cause fatal normal tissue pathology; and MDM2 inhibition can induce p53-dependent cell senescence in mouse fibroblasts. To assess the effects of MI-63 effects in human cells, we compared treatment (2.5-10 uM, 1-7 d) responses in p53wt malignant human mammary epithelial cells (MCF7) with those of p53wt normal human foreskin fibroblasts (HCA2). As expected, MI-63 had little effect on p53mut human cancer cells (MDA-231). However, against p53wt ca...
An attractive therapeutic approach against cancers with wild-type (wt) p53 is the emergent class ... more An attractive therapeutic approach against cancers with wild-type (wt) p53 is the emergent class of small molecule MDM2 inhibitors, including the imidazoline nutlin-3 and more potent spiro-oxindole MI-63 (Ascenta Therapeutics). Both can rapidly increase intracellular p53wt levels and induce apoptosis or a senescence growth arrest, depending on the neoplastic status of the target cell. A critical question in the clinical development of such MDM2 inhibitors is their potential for normal tissue toxicity. In the absence of MDM2, p53 activation can cause fatal normal tissue pathology; and MDM2 inhibition can induce p53-dependent cell senescence in mouse fibroblasts. To assess the effects of MI-63 effects in human cells, we compared treatment (2.5-10 uM, 1-7 d) responses in p53wt malignant human mammary epithelial cells (MCF7) with those of p53wt normal human foreskin fibroblasts (HCA2). As expected, MI-63 had little effect on p53mut human cancer cells (MDA-231). However, against p53wt cancer cells and fibroblasts, MI-63 produced a dose-dependent increase in both nuclear p53 levels and reporter gene (p21) expression within 24 h. MCF7 cells responded with a dose- and time-dependent increase in apoptosis after 2 d exposure (vital dye uptake, cleaved PARP). By contrast, HCA2 cells responded with partially reversible growth arrest and a senescent-like phenotype (increased ROS, SOD2, and SA-βgal) within 3 days. A common early (24 h) MI-63 response by both the malignant and normal cells was nuclear loss of high-mobility group box-1 protein (HMGB1) associated with its active extracellular secretion. Acetylation of this damage-reporting cytokine, thought to be required for its secretion, was detected by western and mass spectrometry analyses. Although HMGB1 is generally pro-inflammatory and may be a harbinger of the senescence-associated secretory phenotype (SASP), MI-63 treated fibroblasts failed to exhibit induction of DNA damage foci or other features characteristic of SASP including IL-6 secretion. Surprisingly, MI-63 treatment of HCA2 beginning 24 or 48 h after a senescence-inducing dose of ionizing radiation (10 Gy) prevented enlarged cell morphology and produced a &gt;80% reduction in SASP associated IL-6 secretion. These findings indicate that upregulation of p53wt in proliferating fibroblasts by MI-63, in the absence of DNA damage or other cell stresses, induces an incomplete senescence like growth arrest without a SASP response, consistent with our finding that p53 restrains SASP. In contrast, MI-63 upregulation of p53wt in malignant epithelial cells caused apoptotic cell death. The early release of HMGB1 from these dying cancer cells may modulate host innate immune response and/or recruit tumor infiltrating inflammatory cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4507. doi:10.1158/1538-7445.AM2011-4507
Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In addition to its well ... more Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In addition to its well established genome stabilizing and tumor suppressing role, p53 can also modulate cell metabolism and mitochondrial function. Small molecule MDM2 antagonists like nutlin-3 or MI-63 (Ascenta) are therapeutic prototypes that rapidly increase wildtype (wt) p53 levels in mammalian cells inducing apoptosis in cancer cells and growth arrest or senescence in non-malignant cells. We employed MI-63 (10 uM × 24h) to induce a near 10-fold increase in p53wt protein expression and compare its mitochondrial bioenergetic and metabolic consequences in malignant (MCF7) and non-malignant (MCF10A) human mammary epithelial cells, whose fates are differentially affected by MI-63. We utilized chromatin immunopreciptation-DNA sequencing (ChIP-seq; Illumina 3G sequencer) and expression microarrays (Affymetrix U133Av2) to identify genome-wide direct and indirect p53 target genes potentially affecting MCF7 mitochondrial structure and function. Within 24h of p53wt upregulation, 1903 genes were differentially expressed including 539 of the 2239 differentially bound direct p53 gene targets, leaving 1364 differentially expressed indirect p53 target genes. While p53 binding was not detected in the mitochondrial genome, GO analysis identified genes associated with the mitochondrial matrix (3 up, 28 down) and genes associated with the outer mitochondrial membrane (7 up, 10 down) as significantly enriched among the p53 regulated nuclear targets (EASE = 0.003, FDR p = 0.03 and EASE = 0.002, FDR p = 0.02 respectively). Real-time, kinetic measurement (Seahorse XF) of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) was performed in adherent cultures of MCF7 and MCF10A cells after comparable 24 h MI-63 induction of p53wt. In contrast to the apparent inhibitory effect of p53wt induction on MCF7 transcription of mitochondrial matrix genes, basal and spare mitochondrial respiratory capacity were stimulated ∼2-fold. This p53wt induced increase in ATP supply and OCR was caused by slightly higher OXPHOS and maximal recruitment of glycolysis, assessed by ECAR and simulated by cell supplementation with pyruvate. Unlike this metabolic and bioenergetic response in the malignant MCF7 cells, p53wt upregulation in the non-malignant MCF10A cells not only failed to stimulate OCR and ECAR but significantly depressed glycolysis. While it is generally thought that the metabolic function of p53wt is to favor energy production by mitochondrial OXPHOS and resist the shift to glycolysis that is characteristically seen in cancer cells, our findings suggest that malignant and non-malignant epithelial cells may have different metabolic and bioenergetic responses to upregulated p53wt. Further studies are needed to determine if these different p53wt responses contribute to the different cell fate responses induced by MDM2 antagonists like MI-63. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3797. doi:10.1158/1538-7445.AM2011-3797
Purpose: We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential... more Purpose: We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential biomarker and therapeutic target in breast cancer. eIF4E facilitates nuclear export and translation of specific, growth-stimulatory mRNAs and is frequently overexpressed in cancer.Experimental Design: Breast cancer cells were treated with ribavirin, an inhibitor of eIF4E, and effects on cell proliferation and on known mRNA targets of eIF4E were determined. eIF4E expression was assessed, at the mRNA and protein level, in breast cancer cell lines and in skin biopsies from patients with metastatic disease. Additionally, pooled microarray data from 621 adjuvant untreated, node-negative breast cancers were analyzed for eIF4E expression levels and correlation with distant metastasis–free survival (DMFS), overall and within each intrinsic breast cancer subtype.Results: At clinically relevant concentrations, ribavirin reduced cell proliferation and suppressed clonogenic potential, correlating w...
Proceedings of the 53rd ACM Technical Symposium on Computer Science Education V. 2, 2022
Past research has shown interactive animations, and those that use real-life analogies in particu... more Past research has shown interactive animations, and those that use real-life analogies in particular, can play an important role in providing the intuition required to understand Computer Science concepts. Nonetheless, the use of analogies continues to be under-explored in CS education compared to other STEM fields. To break the impasse, we aim to create and evaluate a set of interactive animations based on analogies to understand their efficacy. As our first addition, we have created an animation explaining the Buffer Overflow computer systems concept. Explaining the concept abstractly has had a track record of ineffectiveness in our department, since the concept of computer memory as a series of contiguous storage locations is so foreign to students. Instead, the animation uses the analogy of a dry-cleaning shop with a series of hangers to provide a concrete mental picture of computer memory. Students explore various dry-cleaning scenarios, in which customers drop off and pick up their laundry, to understand at their own pace when buffer overflows cause harm and when they are silently ignored. This animation: https://scratch.mit.edu/projects/571317697/ (and others) will be provided as an open educational resource to instructors to encourage the use of interactive analogies in their teaching, and to undergraduate and K-12 students.
Proceedings of the 53rd ACM Technical Symposium on Computer Science Education V. 2, 2022
It is a challenge to engage students when teaching them abstract and complex computer systems con... more It is a challenge to engage students when teaching them abstract and complex computer systems concepts, such as buffer overflow, memory management, concurrent execution, and process synchronization. Past research has shown that interactive animation and real-life analogies make STEM concepts more approachable and help students achieve better learning outcomes. Based on these findings, we introduce interactive analogies into learning the concept of buffer overflow. More specifically, we created a dry-cleaning shop animation tool (https://scratch.mit.edu/projects/571317697/) targeting K-12 and undergraduate students. To assess the effectiveness of our tool, we are in the process of conducting a user study, in which students use our animation tool to learn about buffer overflow and take pre- and post-assessment on the concept. Our goal is to make CS learning more accessible to diverse students, regardless of their background and age.
Cadmium is an environmental contaminant that enters the body through diet or cigarette smoke. It ... more Cadmium is an environmental contaminant that enters the body through diet or cigarette smoke. It affects multiple cellular processes, including cell proliferation, differentiation, and apoptosis. Recently, cadmium has been shown to function as an endocrine disruptor, to stimulate estrogen receptor α (ERα) activity and promote uterine and mammary gland growth in mice. Although cadmium exposure has been associated with the development of breast cancer, the mechanism of action of cadmium remains unclear. To address this deficit, we examined the effects of cadmium treatment on breast cancer cells. We found that ERα is required for both cadmium-induced cell growth and modulation of gene expression. We also determined that ERα translocates to the nucleus in response to cadmium exposure. Additionally, we provide evidence that cadmium potentiates the interaction between ERα and c-Jun and enhances recruitment of this transcription factor complex to the proximal promoters of cyclin D1 and c-m...
Purpose: We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential... more Purpose: We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential biomarker and therapeutic target in breast cancer. eIF4E facilitates nuclear export and translation of specific, growth-stimulatory mRNAs and is frequently overexpressed in cancer. Experimental Design: Breast cancer cells were treated with ribavirin, an inhibitor of eIF4E, and effects on cell proliferation and on known mRNA targets of eIF4E were determined. eIF4E expression was assessed, at the mRNA and protein level, in breast cancer cell lines and in skin biopsies from patients with metastatic disease. Additionally, pooled microarray data from 621 adjuvant untreated, node-negative breast cancers were analyzed for eIF4E expression levels and correlation with distant metastasis–free survival (DMFS), overall and within each intrinsic breast cancer subtype. Results: At clinically relevant concentrations, ribavirin reduced cell proliferation and suppressed clonogenic potential, correlating...
Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In addition to its well ... more Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In addition to its well established genome stabilizing and tumor suppressing role, p53 can also modulate cell metabolism and mitochondrial function. Small molecule MDM2 antagonists like nutlin-3 or MI-63 (Ascenta) are therapeutic prototypes that rapidly increase wildtype (wt) p53 levels in mammalian cells inducing apoptosis in cancer cells and growth arrest or senescence in non-malignant cells. We employed MI-63 (10 uM × 24h) to induce a near 10-fold increase in p53wt protein expression and compare its mitochondrial bioenergetic and metabolic consequences in malignant (MCF7) and non-malignant (MCF10A) human mammary epithelial cells, whose fates are differentially affected by MI-63. We utilized chromatin immunopreciptation-DNA sequencing (ChIP-seq; Illumina 3G sequencer) and expression microarrays (Affymetrix U133Av2) to identify genome-wide direct and indirect p53 target genes potentially affecting MCF7 mitochondrial structure and function. Within 24h of p53wt upregulation, 1903 genes were differentially expressed including 539 of the 2239 differentially bound direct p53 gene targets, leaving 1364 differentially expressed indirect p53 target genes. While p53 binding was not detected in the mitochondrial genome, GO analysis identified genes associated with the mitochondrial matrix (3 up, 28 down) and genes associated with the outer mitochondrial membrane (7 up, 10 down) as significantly enriched among the p53 regulated nuclear targets (EASE = 0.003, FDR p = 0.03 and EASE = 0.002, FDR p = 0.02 respectively). Real-time, kinetic measurement (Seahorse XF) of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) was performed in adherent cultures of MCF7 and MCF10A cells after comparable 24 h MI-63 induction of p53wt. In contrast to the apparent inhibitory effect of p53wt induction on MCF7 transcription of mitochondrial matrix genes, basal and spare mitochondrial respiratory capacity were stimulated ∼2-fold. This p53wt induced increase in ATP supply and OCR was caused by slightly higher OXPHOS and maximal recruitment of glycolysis, assessed by ECAR and simulated by cell supplementation with pyruvate. Unlike this metabolic and bioenergetic response in the malignant MCF7 cells, p53wt upregulation in the non-malignant MCF10A cells not only failed to stimulate OCR and ECAR but significantly depressed glycolysis. While it is generally thought that the metabolic function of p53wt is to favor energy production by mitochondrial OXPHOS and resist the shift to glycolysis that is characteristically seen in cancer cells, our findings suggest that malignant and non-malignant epithelial cells may have different metabolic and bioenergetic responses to upregulated p53wt. Further studies are needed to determine if these different p53wt responses contribute to the different cell fate responses induced by MDM2 antagonists like MI-63. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3797. doi:10.1158/1538-7445.AM2011-3797
Pathways downstream of PI3K/AKT/mTOR that regulate protein translation, ribosome biogenesis, tran... more Pathways downstream of PI3K/AKT/mTOR that regulate protein translation, ribosome biogenesis, transcription, metabolism and autophagy are essential to the formation and progression of many human cancers as evidenced by the clinical approval of such selective mTOR inhibitors as temsirolimus (CCI-779, Wyeth) and everolimus (RAD-001, Novartis). However, clinical limitations to the effective use of mTOR inhibitors include the lack of validated tumor marker(s) predictive of mTOR inhibitor responsiveness, mTOR inhibitor release of S6 kinase-mediated negative feedback resulting in secondary activation of PI3K/AKT, and transcriptional upregulation of the downstream effector eIF4E. Using breast cancer cell lines (MCF7, MCF7/HER2, BT474, SKBr3, MDA231) showing varying sensitivity to RAD-001 (IC50 values for MTT viability @ 5 d from <0.2 nM to >20 nM), we observe marked suppression of phos-p70S6K and secondary phos-AKT activation, with variable suppression of phosSer65-4E-BP1 and downstre...
An attractive therapeutic approach against cancers with wild-type (wt) p53 is the emergent class ... more An attractive therapeutic approach against cancers with wild-type (wt) p53 is the emergent class of small molecule MDM2 inhibitors, including the imidazoline nutlin-3 and more potent spiro-oxindole MI-63 (Ascenta Therapeutics). Both can rapidly increase intracellular p53wt levels and induce apoptosis or a senescence growth arrest, depending on the neoplastic status of the target cell. A critical question in the clinical development of such MDM2 inhibitors is their potential for normal tissue toxicity. In the absence of MDM2, p53 activation can cause fatal normal tissue pathology; and MDM2 inhibition can induce p53-dependent cell senescence in mouse fibroblasts. To assess the effects of MI-63 effects in human cells, we compared treatment (2.5-10 uM, 1-7 d) responses in p53wt malignant human mammary epithelial cells (MCF7) with those of p53wt normal human foreskin fibroblasts (HCA2). As expected, MI-63 had little effect on p53mut human cancer cells (MDA-231). However, against p53wt ca...
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Papers by Rachel Puckett