Katherine Rogers

c-FLIP: A Key Regulator of Colorectal Cancer Cell Death

c-FLIP is an inhibitor of apoptosis mediated by the death receptors Fas, DR4, and DR5 and is expressed as long (c-FLIP(L)) and short (c-FLIP(S)) splice forms. We found that small interfering RNA (siRNA)-mediated silencing of c-FLIP induced spontaneous apoptosis in a panel of p53 wild-type, mutant, and null colorectal cancer cell lines and that this apoptosis was mediated by caspase-8 and Fas-associated death domain. Further analyses indicated the involvement of DR5 and/or Fas (but not DR4) in regulating apoptosis induced by c-FLIP siRNA. Interestingly, these effects were not dependent on activation of DR5 or Fas by their ligands tumor necrosis factor-related apoptosis-inducing ligand and FasL. Overexpression of c-FLIP(L), but not c-FLIP(S), significantly decreased spontaneous and chemotherapy-induced apoptosis in HCT116 cells. Further analyses with splice form-specific siRNAs indicated that c-FLIP(L) was the more important splice form in regulating apoptosis in HCT116, H630, and LoVo cells, although specific knockdown of c-FLIP(S) induced more apoptosis in the HT29 cell line. Importantly, intratumoral delivery of c-FLIP-targeted siRNA duplexes induced apoptosis and inhibited the growth of HCT116 xenografts in BALB/c severe combined immunodeficient mice. In addition, the growth of c-FLIP(L)-overexpressing colorectal cancer xenografts was more rapid than control xenografts, an effect that was significantly enhanced in the presence of chemotherapy. These results indicate that c-FLIP inhibits spontaneous death ligand-independent, death receptor-mediated apoptosis in colorectal cancer cells and that targeting c-FLIP may have therapeutic potential for the treatment of colorectal cancer.

STAT1 A Modulator of Chemotherapy-induced Apoptosis

The anthracyclines, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we showed that these drugs could activate the transcription factor, nuclear factor κB, in a DNA damage-dependent manner. We now show that these drugs can potentiate the activation of signal transducer and activator of transcription 1 (STAT1) in MDA-MB 435 breast cancer cells treated with IFN-γ. We observed that key markers of STAT1 activation, including tyrosine 701 and serine 727 phosphorylation, were enhanced in the presence of doxorubicin. This potentiation resulted in enhanced nuclear localization of activated STAT1 and led to an increase in the nuclear binding of activated STAT complexes. The observed potentiation was specific for STAT1 and IFN-γ, as no effects were observed with either STAT3 or STAT5. Furthermore, the type I IFNs (α and β) had little or no effect. The observed effects on STAT1 phosphorylation have previously been linked with maximal transcriptional activation and apoptosis. Cell viability was assessed by crystal violet staining followed by analysis with CalcuSyn to determine combination index values, a measure of synergy. We confirmed that significant synergy existed between IFN-γ and doxorubicin (combination index = 0.34) at doses lower than IC50 values for this drug (0.67 μmol/L). In support of this, we observed that apoptotic cell death was also enhanced by measuring poly(ADP-ribose) polymerase and caspase-3 cleavage. Finally, suppression of STAT1 expression by small-interfering RNA resulted in a loss of synergistic apoptotic cell death compared with cells, where no suppression of STAT1 expression was attained with scrambled small-interfering RNA control. We conclude that doxorubicin potentiates STAT1 activation in response to IFN-γ, and that this combination results in enhanced apoptosis in breast cancer cells.The anthracyclines, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we showed that these drugs could activate the transcription factor, nuclear factor kappaB, in a DNA damage-dependent manner. We now show that these drugs can potentiate the activation of signal transducer and activator of transcription 1 (STAT1) in MDA-MB 435 breast cancer cells treated with IFN-gamma. We observed that key markers of STAT1 activation, including tyrosine 701 and serine 727 phosphorylation, were enhanced in the presence of doxorubicin. This potentiation resulted in enhanced nuclear localization of activated STAT1 and led to an increase in the nuclear binding of activated STAT complexes. The observed potentiation was specific for STAT1 and IFN-gamma, as no effects were observed with either STAT3 or STAT5. Furthermore, the type I IFNs (alpha and beta) had little or no effect. The observed effects on STAT1 phosphorylation have previously been linked with maximal transcriptional activation and apoptosis. Cell viability was assessed by crystal violet staining followed by analysis with CalcuSyn to determine combination index values, a measure of synergy. We confirmed that significant synergy existed between IFN-gamma and doxorubicin (combination index = 0.34) at doses lower than IC(50) values for this drug (0.67 micromol/L). In support of this, we observed that apoptotic cell death was also enhanced by measuring poly(ADP-ribose) polymerase and caspase-3 cleavage. Finally, suppression of STAT1 expression by small-interfering RNA resulted in a loss of synergistic apoptotic cell death compared with cells, where no suppression of STAT1 expression was attained with scrambled small-interfering RNA control. We conclude that doxorubicin potentiates STAT1 activation in response to IFN-gamma, and that this combination results in enhanced apoptosis in breast cancer cells.

Cellular FLICE-inhibitory protein regulates chemotherapy- induced apoptosis in breast cancer cells

Combination treatment regimens that include topoisomerase-II–targeted drugs, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we showed that IFN-γ and doxorubicin cotreatment synergistically induced apoptosis in MDA435 breast cancer cells in a signal transducer and activator of transcription 1–dependent manner. In this study, we found that this synergy was caspase-8 dependent. In addition, we found that IFN-γ down-regulated the expression of the caspase-8 inhibitor cellular FLICE-inhibitory protein (c-FLIP). Furthermore, IFN-γ down-regulated c-FLIP in a manner that was dependent on the transcription factors signal transducer and activator of transcription 1 and IFN regulatory factor-1. However, IFN-γ had no effect on c-FLIP mRNA levels, indicating that c-FLIP was down-regulated at a posttranscriptional level following IFN-γ treatment. Characterization of the functional significance of c-FLIP modulation by small interfering RNA gene silencing and stable overexpression studies revealed it to be a key regulator of IFN-γ– and doxorubicin-induced apoptosis in MDA435 cells. Analysis of a panel of breast cancer cell lines indicated that c-FLIP was an important general determinant of doxorubicin- and IFN-γ–induced apoptosis in breast cancer cells. Furthermore, c-FLIP gene silencing sensitized MDA435 cells to other chemotherapies, including etoposide, mitoxantrone, and SN-38. These results suggest that c-FLIP plays a pivotal role in modulating drug-induced apoptosis in breast cancer cells. [Mol Cancer Ther 2007;6(5):1544–51]

The role of BRCA1 and BRCA2 mutations in prostate, pancreatic and stomach cancers

The association of germline mutations in the breast cancer susceptibility gene 1 (BRCA1) and the breast cancer susceptibility gene 2 (BRCA2) with the development of breast and ovarian cancers have been widely researched and recognised. It is known that these genes function at multiple sites in the body. Research has subsequently evolved into the connection of BRCA1/2 with cancers at other sites within the body. This review examines the association of BRCA1/2 germline gene mutations with prostate, pancreatic and stomach cancers. An extensive literature search revealed conflicting findings regarding the association of BRCA1/2 gene mutations with these cancers. Most studies suggest that there is an association between BRCA1/2 mutations and carcinoma of the prostate, pancreas and stomach, but some reports propose that such a correlation may be due to factors other than possessing a mutated BRCA1/2 gene, and other associations may be revealed as further epidemiological information becomes available. The review concludes that as more knowledge arises about the mechanisms of BRCA1/2 gene mutations, it should pave the way for future screening programmes to be applied effectively.

The principles and practice of interpreting cardiac rhythm strips: a seven-step model

This open learning zone article examines the cardiac cycle and the interpretation of cardiac rhythm strips. The article begins with a brief revision of related physiology followed by a description of normal sinus rhythm and the main cardiac rhythm abnormalities. The article concludes by providing easy to follow steps for use in the interpretation of cardiac rhythm strips with practice examples presented in the continuing professional development (CPD) task section.

Impact of Enquiry Based Learning (EBL) on Student Midwife Praxis

Midwifery training in Ireland moved to Higher Education in 2006. This shift established a physical and educational separation of theory and practice. The adoption of Enquiry Based Learning (EBL) by one Irish midwifery education institution attempted to address this division. Enquiry Based Learning (EBL) has the potential to develop student reflexivity and evidence assimilation across the career-span and may therefore enhance student praxis. EBL has been championed as an example of an educational model that supports praxis, helping to create competent practitioners through the use of authentic learning scenarios that address the theory practice divide. The current research study represents the first formal evaluation of EBL in undergraduate midwifery education in the South of Ireland. The study was a mixed-methods design that utilised focus groups, interviews and survey to ascertain the opinions of first exposure to EBL amongst a cohort of first year student midwives. Findings demonstrate the value of EBL in enhancing student midwife praxis.

Four steps to interpreting arterial blood gases.

This article examines acid-base balance and the interpretation of arterial blood gases (ABG). The article begins with a brief revision of related physiology, followed by a description of the primary disorders associated with acid-base imbalance. The normal ranges and the significance of abnormal ABG results are explored. The article concludes by providing an easy to follow four-step guide to ABG interpretation with practice examples presented in the CPD task section.

Abstract 2271: Peptidylarginine deiminase 2 as a novel therapeutic target for breast cancer

Peptidylarginine deiminases (PADIs) enzymes are calcium-dependent enzymes that post-translationally convert positively charged protein arginine residues to a neutrally charged citrulline in a process known as citrullination or deimination. This loss of charge can alter the tertiary structure of proteins and affect protein-protein and protein-DNA interactions. We, and others, have found that PADI-mediated histone tail citrullination alters chromatin structure and regulates gene expression. For example, we found that PADI2 interacts with estrogen receptor (ERα) and that PADI2-mediated citrullination of histone H3 arginine 26 residues at ERα binding sites regulates target gene expression. In addition, we also found that PADI2 is often co-expressed with HER2 in both breast cancer cell lines and in tumor tissue. Our molecular studies suggest that PADI2 activates HER2 expression via histone citrullination at the HER2 gene locus and that HER2 signaling appears to induce PADI2 expression, thus potentially forming an oncogenic positive feedback loop with HER2. These findings suggest an important role for PADI2 in breast cancer progression. This prediction is supported by our finding that PADI2 expression is upregulated in cancer cells and that depletion of PADI2 from breast cancer cells results in reduced tumorigenicity. In addition, we found that transgenic overexpression of PADI2 promotes carcinogenesis and that the PADI inhibitor, Cl-Amidine, slows tumor growth in mouse models of breast cancer. Furthermore, we found that Cl-Amidine also maintains basement membrane integrity in xenograft tumors, preventing initiation of metastasis. Together, these findings suggest that PADI2 represents a novel therapeutic target and biomarker for early stage breast cancer. Citation Format: Sachi Horibata, John L. McElwee, David Sadegh, Katherine Rogers, Dalton McLean, Scott A. Coonrod. Peptidylarginine deiminase 2 as a novel therapeutic target for breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2271. doi:10.1158/1538-7445.AM2015-2271

Understanding arterial blood gases

This article examines acid-base balance and the interpretation of arterial blood gases (ABG). The article begins with a brief revision of related physiology followed by a description of the primary disorders of acid-base balance. The normal ranges and the significance of abnormal ABG results are explored. The article concludes by providing an easy to follow four-step guide to ABG interpretation with practice examples presented in the continuing professional development (CPD) task section.