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Phosphorylated cyclic-AMP (cAMP) response element binding protein (p-CREB) is a downstream effector of a variety of important signaling pathways. We investigated whether the human BCRP promoter contains a functional cAMP response element (CRE). 8Br-cAMP, a cAMP analogue, increased the activity of a BCRP promoter reporter construct and BCRP mRNA in human carcinoma cells. Epidermal growth factor receptor (EGFR) pathway activation also led to an increase in p-CREB and in BCRP promoter reporter activity via two major downstream EGFR signaling pathways: the phosphotidylinositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. EGF treatment increased the phosphorylation of EGFR, AKT, ERK and CREB, while simultaneously enhancing BCRP mRNA and functional protein expression. EGF-stimulated CREB phosphorylation and BCRP induction were diminished by inhibition of EGFR, PI3K/AKT or RAS/MAPK signaling. CREB silencing using RNA interference reduced basal levels of BCRP mRNA and diminished the induction of BCRP by EGF. Chromatin immunoprecipitation assays confirmed that a putative CRE site on the BCRP promoter bound p-CREB by a point mutation of the CRE site abolished EGF-induced stimulation of BCRP promoter reporter activity. Furthermore, the CREB co-activator, cAMP-regulated transcriptional co-activator (CRTC2), is involved in CREB-mediated BCRP transcription: androgen depletion of LNCaP human prostate cancer cells increased both CREB phosphorylation and CRTC2 nuclear translocation, and enhanced BCRP expression. Silencing CREB or CRTC2 reduced basal BCRP expression and BCRP induction under androgen-depletion conditions. This novel CRE site plays a central role in mediating BCRP gene expression in several human cancer cell lines following activation of multiple cancer-relevant signaling pathways. [ABSTRACT FROM AUTHOR]

Abstract: Alternative promoter usage is typically associated with mRNAs with differing first exons that contain or consist entirely of a 5′ untranslated region. The murine Bcrp1 (Abcg2) transporter has three alternative promoters associated with mRNAs containing alternative untranslated first exons designated as E1A, E1B, and E1C. The E1B promoter regulates Bcrp1 transcription in mouse intestine. Here, we report the identification and characterization of a novel Bcrp1 promoter and first exon, E1U, located upstream from the other Bcrp1 promoters/first exons, which is the predominant alternative promoter utilized in murine testis. Using in silico analysis we identified a putative steroidogenic factor-1 (SF-1) response element that was unique to the Bcrp1 E1U alternative promoter. Overexpression of SF-1 in murine TM4 Sertoli cells enhanced Bcrp1 E1U mRNA expression and increased Bcrp1 E1U alternative promoter activity in a reporter assay, whereas mutation of the SF-1 binding site totally eliminated Bcrp1 E1U alternative promoter activity. Moreover, expression of Bcrp1 E1U and total mRNA and Bcrp1 protein was markedly diminished in the testes from adult Sertoli cell-specific SF-1 knockout mice, in comparison to the testes from wild-type mice. Binding of SF-1 to the SF-1 response element in the E1U promoter was demonstrated by chromatin immunoprecipitation assays. In conclusion, nuclear transcription factor SF-1 is involved with the regulation of a novel promoter of Bcrp1 that governs transcription of the E1U mRNA isoform in mice. The present study furthers understanding of the complex regulation of Bcrp1 expression in specific tissues of a mammalian model. [Copyright &y& Elsevier]

Abstract: Mouse models are often used to predict drug absorption in humans. Mouse Bcrp1 protein exhibits sequence and functional homology with human BCRP protein. Additionally, BCRP/Bcrp1 expression is regulated by alternative promoter usage in humans and mice; however, the precise intestine-specific alternative promoter utilized in either species is yet to be determined. Therefore we sought to identify and characterize the mouse intestinal Bcrp1 promoter. Using real-time quantitative RT-PCR and 5′ RACE PCR we first established the predominance of a single Bcrp1 first exon (E1b) in the Bcrp1 mRNA isolated throughout the mouse intestine. Simultaneously using 5′ RACE PCR we identified E1C as the predominant BCRP 5′ UTR expressed in the human intestine. Next we established functional activity for the murine promoter upstream of E1b using reporter assays. Subsequently using deletion-construct analysis we found the core promoter region to span −231 to −42bps from the transcriptional start site of E1b. We then predicted a cAMP response element (CRE) as a transcription factor binding site unique only to the E1b promoter region, using in silico methods. We finally established functional interaction of phospho-CREB (p-CREB) protein with the CRE on the E1b promoter using both functional assays and chromatin immunoprecipitation assays. In conclusion, mouse intestinal Bcrp1 expression is regulated by a single alternative promoter upstream of E1b, the predominant Bcrp1 mRNA isoform expressed in the mouse intestine. Furthermore, Bcrp1 E1b mRNA expression is regulated by binding of p-CREB to its cis site on the mouse E1b promoter region. [Copyright &y& Elsevier]

Drug combination is a major treatment approach in cancer and antiviral therapies. A key issue is to find which combinations are additive, synergistic or antagonistic. In this paper, we develop statistical methods for experimental design and data analysis of combination studies of drugs that have log-linear dose-response curves. This class of dose response includes the Hill, sigmoid and simple exponential models. The experimental design (dose-finding and sample size determination) is derived by means of uniform measures that maximize the minimum power of the F-test to detect any departures from additive action and at the same time minimizes the maximum bias due to lack of fit among all potential departures of a given meaningful magnitude. Furthermore, we propose a model-free interaction index surface to capture the interaction of two drugs. The nonparametric function of the interaction index is estimated using the technique developed in thin plate splines. These methods are applicable to both in vivo and in vitro experiments. A study of two anticancer drugs, suberoylanilide hydroxamic acid (Vorinostat) and Etoposide applied sequentially against the cell line HL-60, is given to illustrate the proposed methods of experimental design and interaction analysis. Copyright © 2008 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Flavopiridol is known to modulate the transcription of genes. We investigated the effect of flavopiridol pretreatment on TRAIL cytotoxicity and on the expression of FLIPL in different TRAIL-resistant cell lines, because FLIP expression is known to confer TRAIL-resistance. Apoptosis was assessed by PI staining and protein expression by Western blotting. RT-PCR was used for mRNA quantitation. siRNA gene silencing was used to knock down FLIPL. Flavopiridol pretreatment synergized TRAIL-induced apoptosis in human myeloma and breast cancer cells. Flavopiridol treatment repressed the transcription of FLIPL and downregulated its expression in both myeloma and breast cancer cells. Silencing of FLIPL gene by siRNA sensitized myeloma cells to TRAIL. Flavopiridol treatment downregulated the expression of the proapoptotic members of the Bcl-2 family proteins (Bak, Bax and PUMA-α). The expression of the antiapototic Bcl-2 members (Bcl-2 and Bcl-XL) was not altered by flavopiridol treatment in myeloma cells. Our data indicate that flavopiridol synergizes TRAIL cytotoxicity by downregulation of FLIPL and this synergistic effect is Bcl-2 family independent. [ABSTRACT FROM AUTHOR]

Flavopiridol is known to modulate the transcription of genes. We investigated the effect of flavopiridol pretreatment on TRAIL cytotoxicity and on the expression of FLIPL in different TRAIL-resistant cell lines, because FLIP expression is known to confer TRAIL-resistance. Apoptosis was assessed by PI staining and protein expression by Western blotting. RT-PCR was used for mRNA quantitation. siRNA gene silencing was used to knock down FLIPL. Flavopiridol pretreatment synergized TRAIL-induced apoptosis in human myeloma and breast cancer cells. Flavopiridol treatment repressed the transcription of FLIPL and downregulated its expression in both myeloma and breast cancer cells. Silencing of FLIPL gene by siRNA sensitized myeloma cells to TRAIL. Flavopiridol treatment downregulated the expression of the proapoptotic members of the Bcl-2 family proteins (Bak, Bax and PUMA-α). The expression of the antiapototic Bcl-2 members (Bcl-2 and Bcl-XL) was not altered by flavopiridol treatment in myeloma cells. Our data indicate that flavopiridol synergizes TRAIL cytotoxicity by downregulation of FLIPL and this synergistic effect is Bcl-2 family independent. [ABSTRACT FROM AUTHOR]

Background: In response to the nationwide need for improved care of patients at end of life, our medical school implemented approximately 20 hours of mandatory coursework on the care of dying patients for all students, with satisfactory completion required for graduation. Objective: We now report a long-term evaluation of this coursework. Design/subjects/measurements: A 74-item questionnaire concerning attainment of palliative care and other medical school course objectives, postgraduate practice encounters with patients at end of life, and postgraduate behaviors in palliative care was mailed to all students who graduated from our medical school in 2000, 2001, and 2002. Responses were graded by a Likert-type scale. Additionally, the Association of American Medical Colleges Medical School Graduation Questionnaire (AAMC/GQ) reports for all schools and for our medical school were queried for data regarding palliative care. Results: The return rate of the questionnaire was 54%. The respondents perceived the training to be valuable and effective. They displayed good postgraduate palliative care practices such as choosing to use opiates other than meperidine for pain in the end-of-life setting. Fiftythree percent of the respondents reported that dying patients were often or frequently under their care since graduation. Despite evidence for the efficacy of the training, the respondents perceived that preparedness in certain palliative care domains was somewhat below preparedness in benchmark medical school competencies such as assessment/management of hypertension and diabetes. The AAMC/GQ surveys indicated that for the years 2000–2002, at least 20% more of our graduates perceived that their training in pain management and palliative care was adequate than did all other medical students graduating nationwide. Conclusions: The evaluation provides support for the conclusion that mandatory training in palliative and end-of-life care is effective, perceived to be valuable, and contributes to good palliative and end-of-life care practices in our graduates. Furthermore, the training meets a significant demand in our graduates' clinical practice: end-of-life care. However, expanded medical school emphasis and curriculum hours are still needed for palliative care topics, because preparedness in palliative care skills was perceived to be inferior to benchmark medical skills. To our knowledge, this is the first report of a rigorous summative evaluation of the efficacy of required coursework in palliative and end-of-life care in a U.S. medical school curriculum. [ABSTRACT FROM AUTHOR]

Down-regulation of the androgen receptor (AR) is being evaluated as an effective therapy for the advanced stages of prostate cancer. We report that Ebp1, a protein identified by its interactions with the ErbB3 receptor, down-regulates expression of AR and AR- regulated genes in the LNCaP prostate cancer cell line Using microarray analysis, we identified six endogenous AR target genes, including the AR itself, that are down-regulated by ebp1 overexpression. Chromatin immunoprecipitation assays revealed that Ebp1 was recruited to the prostate-specific antigen gene promoter in response to the androgen antagonist bicalutamide, suggesting that Ebp1 directly affected the expression of AR-regulated genes in response to androgen antagonists. Ebp1 expression was reduced in cells that had become androgen-independent. Androgens failed to stimulate either the growth of ebp1 transfectants or transcription of AR-regulated reporter genes in these cells. The agonist activity of the antiandrogen cyproterone acetate was abolished in ebp1 transfectants. In severe combined immunodeficient mice, Ebp1 overexpression resulted in a reduced incidence of LNCaP tumors and slower tumor growth. These findings suggest that Ebp1 is a previously unrecognized therapeutic target for treatment of hormone refractory prostate cancer. [ABSTRACT FROM AUTHOR]

Our studies demonstrate that the ABC transporter and marker of stem and progenitor cells known as the breast cancer resistance protein (BCRP or ABCG2) confers a strong survival advantage under hypoxic conditions. We show that, under hypoxia, progenitor cells from Bcrp-/- mice have a reduced ability to form colonies as compared with progenitor cells from Bcrp+/+ mice. Blocking BCRP function in Bcrp+/+ progenitor cells markedly reduces survival under hypoxic conditions. However, blocking heme biosynthesis reverses the hypoxic susceptibility of Bcrp-/- progenitor cells, a finding that indicates that heme molecules (i.e. porphyrins) are detrimental to Bcrp-/- cells under hypoxia. BCRP specifically binds heme, and cells lacking BCRP accumulate porphyrins. Finally, Bcrp expression is up-regulated by hypoxia, and we demonstrate that this up-regulation involves the hyopoxia-inducible transcription factor complex HIF-1. Collectively, our findings suggest that cells can, upon hypoxic demand, use BCRP to reduce heme or porphyrin accumulation, which can be detrimental to cells. Our findings have implications for the survival of stem cells and tumor cells in hypoxic environments. [ABSTRACT FROM AUTHOR]

Background. The need for better care for terminally ill patients led us to create an educational program to provide internal medicine residents and medical oncology fellows basic competency in palliative and end-of-life care. Methods. An interdisciplinary team identified educational strategies, course objectives, content, and evaluation instruments. Results and Conclusions. Our strategy is to use a required Web-based course to establish a knowledge base upon which specific training during clinical rotations build skills. Field testing of the Web course showed it was an effective tool for delivering clinically applicable content. Skill building experiences are now being integrated into selected clinical rotations. [ABSTRACT FROM AUTHOR]

Stimulated by support from an R25 grant from the National Cancer Institute, we assembled a multidisciplinary team to design, implement, evaluate, and institutionalize in our medical school curriculum a training program to enable all students to graduate with basic competency in palliative and end-of-life care. In the fall of 1994, we reviewed the medical curriculum extensively to determine the need and optimal sites for integration of new educational modules. The freshman and junior years were found most suitable for our purposes; hence, behavioral objectives targeted at the freshman and junior medical student as learner were designed for the domain of palliative and end-of-life care. By 1996, negotiations with the medical school curriculum committee and with individual course masters secured permission and time allotments to include new coursework as required elements. We integrated approximately 20 hours of required training into the medical school curriculum, accomplished by modules that utilized small group learning, didactic presentations, out-of-classroom assignments, and practicum/experiential teaching methods. Freshman year curriculum consisted of a 3-hour module that considers the role of the physician in end-of-life care. Junior year training included a 1-hour module on advance directives and a 16-hour classroom and practicum course on hospice and end-of-life care. Performance-based assessment revealed that the students achieved the behavioral objectives. Furthermore, the students perceived benefit from the training and concluded that palliative care education should be a required part of their medical school experience. In September 2000, the medical school's Clinical Years Committee officially designated the palliative and end-of-life care training modules a mandatory part of the curriculum, with satisfactory completion a requirement for graduation. [ABSTRACT FROM AUTHOR]

Physical symptoms other than pain often contribute to suffering near the end of life. In addition to pain, the most common symptoms in the terminal stages of an illness such as cancer or acquired immunodeficiency syndrome are fatigue, anorexia, cachexia, nausea, vomiting, constipation, delirium and dyspnea. Management involves a diagnostic evaluation for the cause of each symptom when possible, treatment of the identified cause when reasonable, and concomitant treatment of the symptom using nonpharmacologic and adjunctive pharmacologic measures. Part I of this two-part article discusses fatigue, anorexia, cachexia, nausea and vomiting. Fatigue is the most common symptom at the end of life, but little is known about its pathophysiology and specific treatment. Education of the patient and family is the foundation of treatment, with the possible use of adjunctive psychostimulants. Anorexia and cachexia caused by wasting syndromes are best managed with patient and family education, as well as a possible trial of appetite stimulants such as megestrol or dexamethasone. For appropriate pharmacologic treatment, it is helpful to identify the pathophysiologic origin of nausea in each patient. [ABSTRACT FROM AUTHOR]

Describes an adult education program that was developed from a general palliative medicine curriculum. Modification to nursing facility (NF) physician curriculum; Goals for NF physicians from a general medical student curricula.

The article presents a case study of a 66-year-old man who was diagnosed with concurrent myelodysplastic syndrome (MDS) and multiple myeloma (MM) in the bone marrow (BM). Several diagnostic tests were conducted to determine his condition including single nucleotide polymorphism (SNP) genotyping microarray and fluorescence in situ hybridization (FISH). It demonstrates the importance of comprehensive morphologic, cytogenetic, and gene array tests in the correct diagnosis of the disease.

Simple Summary: In the present study, we sought to determine whether a commonly used oral drug to treat adult-onset diabetes, metformin, which has a longstanding clinical history and known safety and tolerability profile, can improve the anti-cancer effects of two well-established oral agents currently in use to treat advanced prostate cancer, abiraterone and enzalutamide. We used androgen-sensitive cell culture models of human prostate cancer to test our hypothesis. We found that metformin and the oral anti-prostate cancer agents together are more effective in inhibiting prostate cancer cell growth and inducing prostate cancer cell death than when used alone. We identified new pathways by which the enhanced anti-cancer effects occur with the combination treatments. The present work suggests that incorporating metformin with abiraterone or enzalutamide may improve treatment outcomes in hormone sensitive prostate cancer. We explored whether the anti-prostate cancer (PC) activity of the androgen receptor-axis-targeted agents (ARATs) abiraterone and enzalutamide is enhanced by metformin. Using complementary biological and molecular approaches, we determined the associated underlying mechanisms in pre-clinical androgen-sensitive PC models. ARATs increased androgren receptors (ARs) in LNCaP and AR/ARv7 (AR variant) in VCaP cells, inhibited cell proliferation in both, and induced poly(ADP-ribose) polymerase-1 (PARP-1) cleavage and death in VCaP but not LNCaP cells. Metformin decreased AR and ARv7 expression and induced cleaved PARP-1-associated death in both cell lines. Metformin with abiraterone or enzalutamide decreased AR and ARv7 expression showed greater inhibition of cell proliferation and greater induction of cell death than single agent treatments. Combination treatments led to increased cleaved PARP-1 and enhanced PARP-1 activity manifested by increases in poly(ADP-ribose) (PAR) and nuclear accumulation of apoptosis inducing factor (AIF). Enhanced annexin V staining occurred in LNCaP cells only with metformin/ARAT combinations, but no caspase 3 recruitment occurred in either cell line. Finally, metformin and metformin/ARAT combinations increased lysosomal permeability resulting in cathepsin G-mediated PARP-1 cleavage and cell death. In conclusion, metformin enhances the efficacy of abiraterone and enzalutamide via two PARP-1-dependent, caspase 3-independent pathways, providing a rationale to evaluate these combinations in castration-sensitive PC. [ABSTRACT FROM AUTHOR]

Discusses several issues related to the American Association for Cancer Education (AACE) in the U.S. Success of the interim Executive Council meeting in February 2005; Information on the 2005 AACE Annual Meeting in Cincinnati, Ohio; Appointment of several association members to update the AACE Web site; Kickoff of a membership drive.

The article presents the author's parting remarks as the outgoing president of the American Association for Cancer Education (AACE). The author expresses how he feels as his term formally ended in December 2005. The challenges and responsibilities of serving as an AACE officer are described.