Early Cancer Research Education for Underrepresented Middle School Students: A Case Study of Experiences from Youth Enjoy Science Programs.

Cancer is the second leading cause of death in the United States, and marginalized communities are disproportionately affected. There is a significant need to address cancer disparities and the determinants of health that are associated with those disparities. Increasing the diversity of the cancer research workforce is a potential mechanism to address health disparities. The National Cancer Institute's Youth Enjoy Science Research Education Program provides funding to engage middle school, high school, and undergraduate students from underrepresented student populations in cancer research education programming, conduct cancer education outreach to communities, and provide cancer research curricula to kindergarten through grade 12 educators. The ultimate goal of these programs is to motivate, prepare, and support students in pursuing cancer research careers. Herein, we describe how three academic institutions with YES Programs - the University of Kentucky, the University of Nebraska Medical Center, and Case Western Reserve University - provide cancer education programming to middle school students. Descriptions of each institutions' programing are provided. Common and unique elements were determined through an evaluation of the three programs. Although each program was developed independently, they have more common than unique elements. We provide insight into the development of middle school programs for other academic institutions.

Teach to Beat Cancer: An Integral Component of the Case Comprehensive Cancer Center Youth Enjoy Science Program.

The Youth Enjoy Science -Teach To Beat Cancer program funded by the National Cancer Institute utilizes the resources of the Case Western Reserve University School of Medicine and Case Comprehensive Cancer Center to provide experiences and training for grade 6-12 teachers so that they are expertly equipped to develop curricular approaches to take back to their classrooms to enhance science education, introduce concepts of cancer biology, encourage cancer risk reduction activities, foster disparity elimination and to motivate middle and high school students to pursue careers in biomedical sciences and cancer research. The program focuses on four aspects of teacher engagement and education: 1) Research Engagement, 2) Curriculum Development, 3) Risk Reduction and Disparity Elimination, and 4) Advocacy for Cancer Research and Cancer Research Careers. This program has been crucial to achieve the National Cancer Institute's goal of educating students from diverse backgrounds underrepresented in biomedical research in the Cleveland area as each teacher influences a significant number of students in their classrooms each year, and are introducing students to cancer biology, exciting them to consider careers in cancer prevention, diagnosis, control, treatment, and research. This article provides an overview of the program including its impact on the teachers and their students.

Disparities Education Strategies in the Case Comprehensive Cancer Center STEM Focused Youth Enjoy Science Program.

The Youth Enjoy Science/Scientific Enrichment and Opportunity (YES/SEO) Program at Case Western Reserve University (CWRU) School of Medicine and the Case Comprehensive Cancer Center (Case CCC) in Cleveland, OH is an intensive Research, Science, Technology, Engineering, and Mathematics (STEM) program targeted at engaging underrepresented minority high school students to better understand and to pursue careers in cancer research and healthcare. The program's long-range goals are to increase diversity of the cancer professional workforce to contribute to elimination of cancer health inequities. A challenging aspect of this intensive research and STEM education program is how to effectively teach about cancer health disparities and to address the importance of developing strategies for their remediation. We describe herein some innovative approaches utilized to engage students in learning about disparities and thinking about solutions. Overall, feedback from our students indicates the importance of introducing disparities education topics often and using multiple approaches, including small and large meetings as well as lecture and conversational formats. These approaches provide opportunities for frequent student engagement and concept reinforcement. Based on this experience, a series of recommendations are provided for incorporating disparities education into intensive research and STEM programs.

Sustaining Student Engagement - Successes and Challenges of a Virtual STEM Program for High School Students.

Case Western Reserve University's School of Medicine and Comprehensive Cancer Center coordinate in-depth research immersion STEM programs to engage high school students in biomedical research and encourage pursuit of careers in health-related research and clinical care. Due to COVID-19, the 2020 programs were delivered entirely virtually. Student and faculty perceptions of the virtual experience were evaluated using surveys and focus groups. Ninety percent of students felt the virtual program met expectations. Student rankings for programmatic components that could remain virtual in future years showed a preference for highly interactive activities, especially mentorship and dialogue-based activities like discussions of science in the news. Ninety-seven percent of faculty agreed students' scientific knowledge improved. Faculty commented that certain research projects (e.g., data analysis, literature reviews) were highly appropriate for a virtual program, but that the lack of hands-on laboratory activities was challenging. Increased individual attention, flexibility, and independence were hailed as strengths of the virtual program. These findings identify activities that sustain student interest in biomedical, healthcare, and cancer related research using a virtual medium and indicate mentorship and interactive discussion-based activities enhance virtual education. Moreover, the results support incorporation of interactive online pedagogical approaches to enhance student engagement virtually and in-person.

Youth Enjoy Science Program at the Case Comprehensive Cancer Center: Increasing Engagement and Opportunity for Underrepresented Minority Students.

The Youth Enjoy Science (YES) Program at the Case Comprehensive Cancer Center is a National Cancer Institute (NCI) R25-funded training grant, designed to increase the pipeline of underrepresented minority (URM) students entering college and pursuing biomedical research and health care careers in the Cleveland Metropolitan and surrounding school districts. The three components of the program include: Learn to Beat Cancer, engaging middle school students and their families; Research to Beat Cancer, designed for high school students and college undergraduates; and Teach to Beat Cancer, focused on enhancing science, technology, engineering, and mathematics (STEM) teaching capacity among high school teachers. This study focuses on Research to Beat Cancer, which, in 2018 enrolled 36 URM students as paid summer scholars. Students were assigned to a faculty mentor, were taught laboratory safety, responsible conduct of research and the scientific method, and then immersed in full-time laboratory cancer research during an eight-week period. Twice each week, students participated in Lunch and Learn Seminars where faculty members provided combined motivational and scientific guidance lectures. In a capstone poster session at the end of the program, students presented their research to peers, medical and graduate students, family members, faculty, community members and leaders. Students' perceptions of the program were reported using descriptive statistics and qualitative thematic analyses. Twenty-four of the 2018 YES students (67%) and 19 (53%) mentors completed the online post-program survey. Opportunity was a major qualitative theme from student and mentor responses. Future research will investigate the long-term impacts of YES, including college enrollment.

The opposing effects of interferon-beta and oncostatin-M as regulators of cancer stem cell plasticity in triple-negative breast cancer.

BACKGROUND: Highly aggressive, metastatic and therapeutically resistant triple-negative breast cancers (TNBCs) are often enriched for cancer stem cells (CSC). Cytokines within the breast tumor microenvironment (TME) influence the CSC state by regulating tumor cell differentiation programs. Two prevalent breast TME cytokines are oncostatin-M (OSM) and interferon-ß (IFN-ß). OSM is a member of the IL-6 family of cytokines and can drive the de-differentiation of TNBC cells to a highly aggressive CSC state. Conversely, IFN-ß induces the differentiation of TNBC, resulting in the repression of CSC properties. Here, we assess how these breast TME cytokines influence CSC plasticity and clinical outcome. METHODS: Using transformed human mammary epithelial cell (HMEC) and TNBC cell models, we assessed the CSC markers and properties following exposure to OSM and/or IFN-ß. CSC markers included CD24, CD44, and SNAIL; CSC properties included tumor sphere formation, migratory capacity, and tumor initiation. RESULTS: There are three major findings from our study. First, exposure of purified, non-CSC to IFN-ß prevents OSM-mediated CD44 and SNAIL expression and represses tumor sphere formation and migratory capacity. Second, during OSM-induced de-differentiation, OSM represses endogenous IFN-ß mRNA expression and autocrine/paracrine IFN-ß signaling. Restoring IFN-ß signaling to OSM-driven CSC re-engages IFN-ß-mediated differentiation by repressing OSM/STAT3/SMAD3-mediated SNAIL expression, tumor initiation, and growth. Finally, the therapeutic use of IFN-ß to treat OSM-driven tumors significantly suppresses tumor growth. CONCLUSIONS: Our findings suggest that the levels of IFN-ß and OSM in TNBC dictate the abundance of cells with a CSC phenotype. Indeed, TNBCs with elevated IFN-ß signaling have repressed CSC properties and a better clinical outcome. Conversely, TNBCs with elevated OSM signaling have a worse clinical outcome. Likewise, since OSM suppresses IFN-ß expression and signaling, our studies suggest that strategies to limit OSM signaling or activate IFN-ß signaling will disengage the de-differentiation programs responsible for the aggressiveness of TNBCs.

Interferon-beta represses cancer stem cell properties in triple-negative breast cancer.

Triple-negative breast cancer (TNBC), the deadliest form of this disease, lacks a targeted therapy. TNBC tumors that fail to respond to chemotherapy are characterized by a repressed IFN/signal transducer and activator of transcription (IFN/STAT) gene signature and are often enriched for cancer stem cells (CSCs). We have found that human mammary epithelial cells that undergo an epithelial-to-mesenchymal transition (EMT) following transformation acquire CSC properties. These mesenchymal/CSCs have a significantly repressed IFN/STAT gene expression signature and an enhanced ability to migrate and form tumor spheres. Treatment with IFN-beta (IFN-ß) led to a less aggressive epithelial/non-CSC-like state, with repressed expression of mesenchymal proteins (VIMENTIN, SLUG), reduced migration and tumor sphere formation, and reexpression of CD24 (a surface marker for non-CSCs), concomitant with an epithelium-like morphology. The CSC-like properties were correlated with high levels of unphosphorylated IFN-stimulated gene factor 3 (U-ISGF3), which was previously linked to resistance to DNA damage. Inhibiting the expression of IRF9 (the DNA-binding component of U-ISGF3) reduced the migration of mesenchymal/CSCs. Here we report a positive translational role for IFN-ß, as gene expression profiling of patient-derived TNBC tumors demonstrates that an IFN-ß metagene signature correlates with improved patient survival, an immune response linked with tumor-infiltrating lymphocytes (TILs), and a repressed CSC metagene signature. Taken together, our findings indicate that repressed IFN signaling in TNBCs with CSC-like properties is due to high levels of U-ISGF3 and that treatment with IFN-ß reduces CSC properties, suggesting a therapeutic strategy to treat drug-resistant, highly aggressive TNBC tumors.

STAT3-mediated SMAD3 activation underlies Oncostatin M-induced Senescence.

Cytokines in the developing tumor microenvironment (TME) can drive transformation and subsequent progression toward metastasis. Elevated levels of the Interleukin-6 (IL-6) family cytokine Oncostatin M (OSM) in the breast TME correlate with aggressive, metastatic cancers, increased tumor recurrence, and poor patient prognosis. Paradoxically, OSM engages a tumor-suppressive, Signal Transducer and Activator of Transcription 3 (STAT3)-dependent senescence response in normal and non-transformed human mammary epithelial cells (HMEC). Here, we identify a novel link between OSM-activated STAT3 signaling and the Transforming Growth Factor-ß (TGF-ß) signaling pathway that engages senescence in HMEC. Inhibition of functional TGF-ß/SMAD signaling by expressing a dominant-negative TGF-ß receptor, treating with a TGF-ß receptor inhibitor, or suppressing SMAD3 expression using a SMAD3-shRNA prevented OSM-induced senescence. OSM promoted a protein complex involving activated-STAT3 and SMAD3, induced the nuclear localization of SMAD3, and enhanced SMAD3-mediated transcription responsible for senescence. In contrast, expression of MYC (c-MYC) from a constitutive promoter abrogated senescence and strikingly, cooperated with OSM to promote a transformed phenotype, epithelial-mesenchymal transition (EMT), and invasiveness. Our findings suggest that a novel STAT3/SMAD3-signaling axis is required for OSM-mediated senescence that is coopted during the transformation process to confer aggressive cancer cell properties. Understanding how developing cancer cells bypass OSM/STAT3/SMAD3-mediated senescence may help identify novel targets for future "pro-senescence" therapies aiming to reengage this hidden tumor-suppressive response.

Cancer Stem Cell Plasticity Drives Therapeutic Resistance.

The connection between epithelial-mesenchymal (E-M) plasticity and cancer stem cell (CSC) properties has been paradigm-shifting, linking tumor cell invasion and metastasis with therapeutic recurrence. However, despite their importance, the molecular pathways involved in generating invasive, metastatic, and therapy-resistant CSCs remain poorly understood. The enrichment of cells with a mesenchymal/CSC phenotype following therapy has been interpreted in two different ways. The original interpretation posited that therapy kills non-CSCs while sparing pre-existing CSCs. However, evidence is emerging that suggests non-CSCs can be induced into a transient, drug-tolerant, CSC-like state by chemotherapy. The ability to transition between distinct cell states may be as critical for the survival of tumor cells following therapy as it is for metastatic progression. Therefore, inhibition of the pathways that promote E-M and CSC plasticity may suppress tumor recurrence following chemotherapy. Here, we review the emerging appreciation for how plasticity confers therapeutic resistance and tumor recurrence.

Cancer stem cells: targeting the roots of cancer, seeds of metastasis, and sources of therapy resistance.

With the goal to remove the roots of cancer, eliminate metastatic seeds, and overcome therapy resistance, the 2014 inaugural International Cancer Stem Cell (CSC) Conference at Cleveland, OH, convened together over 320 investigators, including 55 invited world-class speakers, 25 short oral presenters, and 100 poster presenters, to gain an in-depth understanding of CSCs and explore therapeutic opportunities targeting CSCs. The meeting enabled intriguing discussions on several topics including: genetics and epigenetics; cancer origin and evolution; microenvironment and exosomes; metabolism and inflammation; metastasis and therapy resistance; single cell and heterogeneity; plasticity and reprogramming; as well as other new concepts. Reports of clinical trials targeting CSCs emphasized the urgent need for strategically designing combinational CSC-targeting therapies against cancer.

HiJAK'd Signaling; the STAT3 Paradox in Senescence and Cancer Progression.

Clinical and epidemiological data have associated chronic inflammation with cancer progression. Most tumors show evidence of infiltrating immune and inflammatory cells, and chronic inflammatory disorders are known to increase the overall risk of cancer development. While immune cells are often observed in early hyperplastic lesions in vivo, there remains debate over whether these immune cells and the cytokines they produce in the developing hyperplastic microenvironment act to inhibit or facilitate tumor development. The interleukin-6 (IL-6) family of cytokines, which includes IL-6 and oncostatin M (OSM), among others (LIF, CT-1, CNTF, and CLC), are secreted by immune cells, stromal cells, and epithelial cells, and regulate diverse biological processes. Each of the IL-6 family cytokines signals through a distinct receptor complex, yet each receptor complex uses a shared gp130 subunit, which is critical for signal transduction following cytokine binding. Activation of gp130 results in the activation of Signal Transducer and Activator of Transcription 3 (STAT3), and the Mitogen-Activated Protein Kinase (MAPK) and Phosphatidylinositol 3-Kinase (PI3K) signaling cascades. Tumor suppressive signaling can often be observed in normal cells following prolonged STAT3 activation. However, there is mounting evidence that the IL-6 family cytokines can contribute to later stages of tumor progression in many ways. Here we will review how the microenvironmental IL-6 family cytokine OSM influences each stage of the transformation process. We discuss the intrinsic adaptations a developing cancer cell must make in order to tolerate and circumvent OSM-mediated growth suppression, as well as the OSM effectors that are hijacked during tumor expansion and metastasis. We propose that combining current therapies with new ones that suppress the signals generated from the tumor microenvironment will significantly impact an oncologist's ability to treat cancer.

Tumor microenvironmental signaling elicits epithelial-mesenchymal plasticity through cooperation with transforming genetic events.

Epithelial-to-mesenchymal transition (EMT) facilitates the escape of epithelial cancer cells from the primary tumor site, which is a key event early in metastasis. Here, we explore how extrinsic, tumor microenvironmental cytokines cooperate with intrinsic, genetic changes to promote EMT in human mammary epithelial cells (HMECs). Viral transduction of transforming genetic events into HMECs routinely generated two distinct cell populations. One population retained epithelial characteristics, while an emergent population spontaneously acquired a mesenchymal morphology and properties associated with cancer stem cells (CSCs). Interestingly, the spontaneous mesenchymal/CSCs were unable to differentiate and lacked epithelial-mesenchymal plasticity. In contrast, exposure of the transformed HMECs retaining epithelial characteristics to exogenous transforming growth factor-ß (TGF-ß) generated a mesenchymal/CSC population with remarkable plasticity. The TGF-ß-induced mesenchymal/CSC population was dependent on the continued presence of TGF-ß. Removal of TGF-ß or pharmacologic or genetic inhibition of TGF-ß/SMAD signaling led to the reversion of mesenchymal/CSC to epithelial/non-CSC. Our results demonstrate that targeting exogenous cytokine signaling disrupts epithelial-mesenchymal plasticity and may be an effective strategy to inhibit the emergence of circulating tumor cells. The model of epithelial-mesenchymal plasticity we describe here can be used to identify novel tumor microenvironmental factors and downstream signaling that cooperate with intrinsic genetic changes to drive metastasis. Understanding the interaction between extrinsic and intrinsic factors that regulate epithelial-mesenchymal plasticity will allow the development of new therapies that target tumor microenvironmental signals to reduce metastasis.

IFNß-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage.

A single high dose of interferon-ß (IFNß) activates powerful cellular responses, in which many anti-viral, pro-apoptotic, and anti-proliferative proteins are highly expressed. Since some of these proteins are deleterious, cells downregulate this initial response rapidly. However, the expression of many anti-viral proteins that do no harm is sustained, prolonging a substantial part of the initial anti-viral response for days and also providing resistance to DNA damage. While the transcription factor ISGF3 (IRF9 and tyrosine-phosphorylated STATs 1 and 2) drives the first rapid response phase, the related factor un-phosphorylated ISGF3 (U-ISGF3), formed by IFNß-induced high levels of IRF9 and STATs 1 and 2 without tyrosine phosphorylation, drives the second prolonged response. The U-ISGF3-induced anti-viral genes that show prolonged expression are driven by distinct IFN stimulated response elements (ISREs). Continuous exposure of cells to a low level of IFNß, often seen in cancers, leads to steady-state increased expression of only the U-ISGF3-dependent proteins, with no sustained increase in other IFNß-induced proteins, and to constitutive resistance to DNA damage.

Constitutive CCND1/CDK2 activity substitutes for p53 loss, or MYC or oncogenic RAS expression in the transformation of human mammary epithelial cells.

Cancer develops following the accumulation of genetic and epigenetic alterations that inactivate tumor suppressor genes and activate proto-oncogenes. Dysregulated cyclin-dependent kinase (CDK) activity has oncogenic potential in breast cancer due to its ability to inactivate key tumor suppressor networks and drive aberrant proliferation. Accumulation or over-expression of cyclin D1 (CCND1) occurs in a majority of breast cancers and over-expression of CCND1 leads to accumulation of activated CCND1/CDK2 complexes in breast cancer cells. We describe here the role of constitutively active CCND1/CDK2 complexes in human mammary epithelial cell (HMEC) transformation. A genetically-defined, stepwise HMEC transformation model was generated by inhibiting p16 and p53 with shRNA, and expressing exogenous MYC and mutant RAS. By replacing components of this model, we demonstrate that constitutive CCND1/CDK2 activity effectively confers anchorage independent growth by inhibiting p53 or replacing MYC or oncogenic RAS expression. These findings are consistent with several clinical observations of luminal breast cancer sub-types that show elevated CCND1 typically occurs in specimens that retain wild-type p53, do not amplify MYC, and contain no RAS mutations. Taken together, these data suggest that targeted inhibition of constitutive CCND1/CDK2 activity may enhance the effectiveness of current treatments for luminal breast cancer.

p53 induces distinct epigenetic states at its direct target promoters.

BACKGROUND: The tumor suppressor protein p53 is a transcription factor that is mutated in many cancers. Regulation of gene expression by binding of wild-type p53 to its target sites is accompanied by changes in epigenetic marks like histone acetylation. We studied DNA binding and epigenetic changes induced by wild-type and mutant p53 in non-malignant hTERT-immortalized human mammary epithelial cells overexpressing either wild-type p53 or one of four p53 mutants (R175H, R249S, R273H and R280K) on a wild-type p53 background. RESULTS: Using chromatin immunoprecipitation coupled to a 13,000 human promoter microarray, we found that wild-type p53 bound 197 promoters on the microarray including known and novel p53 targets. Of these p53 targets only 20% showed a concomitant increase in histone acetylation, which was linked to increased gene expression, while 80% of targets showed no changes in histone acetylation. We did not observe any decreases in histone acetylation in genes directly bound by wild-type p53. DNA binding in samples expressing mutant p53 was reduced over 95% relative to wild-type p53 and very few changes in histone acetylation and no changes in DNA methylation were observed in mutant p53 expressing samples. CONCLUSION: We conclude that wild-type p53 induces transcription of target genes by binding to DNA and differential induction of histone acetylation at target promoters. Several new wild-type p53 target genes, including DGKZ, FBXO22 and GDF9, were found. DNA binding of wild-type p53 is highly compromised if mutant p53 is present due to interaction of both p53 forms resulting in no direct effect on epigenetic marks.

Different mutant/wild-type p53 combinations cause a spectrum of increased invasive potential in nonmalignant immortalized human mammary epithelial cells.

Aberrations of p53 occur in most, if not all, human cancers. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Immortalized human mammary epithelial cells resemble the earliest forms of aberrant breast tissue growth but do not express many malignancy-associated phenotypes. We created a model of human mammary epithelial tumorigenesis by infecting hTERT-HME1 immortalized human mammary epithelial cells expressing wild-type p53 with four different mutant p53 constructs to determine the role of p53 mutation on the evolution of tumor phenotypes. We demonstrate that different mutant/wild-type p53 heterozygous models generate loss of function, dominant negative activity, and a spectrum of gain of function activities that induce varying degrees of invasive potential. We suggest that this model can be used to elucidate changes that occur in early stages of human mammary epithelial tumorigenesis. These changes may constitute novel biomarkers or reveal novel treatment modalities that could inhibit progression from primary to metastatic breast disease.

Epigenetic silencing of DSC3 is a common event in human breast cancer.

INTRODUCTION: Desmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens. METHODS: We used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines. RESULTS: DSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure. CONCLUSION: These results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure.

The acetyltransferase p300/CBP-associated factor is a p53 target gene in breast tumor cells.

p300/CBP-associated factor (PCAF) is a coactivator of the tumor suppressor, p53. PCAF participates in p53's transactivation of target genes through acetylation of both bound p53 and histones within p53 target promoters. Using microarrays, we discovered that PCAF itself is induced by p53 in a panel of breast tumor cell lines. Two p53 mutant breast tumor cell lines, BT-549 and UACC-1179, were chosen for further study of PCAF induction by wild-type p53. PCAF induction following adenoviral transduction of p53 expression was confirmed with real-time polymerase chain reaction in a time course experiment. Chromatin immunoprecipitation experiments then showed that PCAF induction was associated with increased p53 binding to the PCAF promoter, which contains p53 consensus-binding sites. PCAF induction by p53 activity was further demonstrated in wild-type p53 MCF10A cells when PCAF expression was induced following activation of endogenous wild-type p53 with doxorubicin in a dose- and time-dependent manner. Furthermore, the doxorubicin-induced increase in PCAF expression was blocked by pretreatment of the MCF10A cells with siRNA (small interfering RNA) targeted against p53 mRNA. Taken together, the results show that PCAF expression can be induced by wild-type p53.

Mutant p53 and aberrant cytosine methylation cooperate to silence gene expression.

p53 is an important transcriptional regulator that is frequently mutated in cancer. Gene-profiling experiments of breast cancer cells infected with wt p53 revealed both MASPIN and desmocollin 3 (DSC3) to be p53-target genes, even though both genes are silenced in association with aberrant cytosine methylation of their promoters. Despite the transcriptional repression of these genes by aberrant DNA methylation, restoration of p53 resulted in the partial reactivation of both genes. This reactivation is a result of wt p53 binding to its consensus DNA-binding sites within the MASPIN and DSC3 promoters, stimulating histone acetylation, and enhancing chromatin accessibility of their promoters. Interestingly, wt p53 alone did not affect the methylation status of either promoter, suggesting that p53 itself can partially overcome the repressive barrier of DNA methylation. Pharmacologic inhibition of DNA methylation with 5-aza-2'-deoxycytidine in combination with restoration of wt p53 status resulted in a synergistic reactivation of these genes to near-normal levels. These results suggest that cancer treatments that target both genetic and epigenetic facets of gene regulation may be a useful strategy towards the therapeutic transcriptional reprogramming of cancer cells.

Isolation and expression analysis of the isopropylmalate synthase gene family of Arabidopsis thaliana.

Isopropylmalate synthase (IPMS) is the first enzyme in the leucine biosynthetic pathway. It is the branch point in the biosynthesis of leucine and the other branched-chain amino acids. IPMS is also regulated by negative feedback inhibition by the end-product leucine. There are four highly homologous loci within the Arabidopsis thaliana genome, which contain sequences that code for IPMS. Through library screening and RT-PCR the expression patterns of three of these loci namely IMS1, IMS2, and IMS3 have been isolated and then characterized. cDNAs of IMS2 and IMS3 lacking the 5' chloroplast leader sequence were able to complement a leucine auxotroph of E. coli.