Stable Differences in Intrinsic Mitochondrial Membrane Potential of Tumor Cell Subpopulations Reflect Phenotypic Heterogeneity

Heterogeneity among cells that constitute a solid tumor is important in determining disease progression. Our previous work established that, within a population of metastatic colonic tumor cells, there are minor subpopulations of cells with stable differences in their intrinsic mitochondrial membrane potential (ΔΨm), and that these differences in ΔΨm are linked to tumorigenic phenotype. Here we expanded this work to investigate primary mammary, as well as colonic, tumor cell lines. We show that within a primary mammary tumor cell population, and in both primary and metastatic colonic tumor cell populations, there are subpopulations of cells with significant stable variations in intrinsic ΔΨm. In each of these 3 tumor cell populations, cells with relatively higher intrinsic ΔΨm exhibit phenotypic properties consistent with promotion of tumor cell survival and expansion. However, additional properties associated with invasive potential appear in cells with higher intrinsic ΔΨm only from the metastatic colonic tumor cell line. Thus, it is likely that differences in the intrinsic ΔΨm among cells that constitute primary mammary tumor populations, as well as primary and metastatic colonic tumor populations, are markers of an acquired tumor phenotype which, within the context of the tumor, influence the probability that particular cells will contribute to disease progression

ELISA in the multiplex era: potentials and pitfalls

Multiplex immunoassays confer several advantages over widely adopted singleplex immunoassays including increased efficiency at a reduced expense, greater output per sample volume ratios and higher throughput predicating more resolute, detailed diagnostics and facilitating personalised medicine. Nonetheless, to date, relatively few protein multiplex immunoassays have been validated for in vitro diagnostics in clinical/point-of-care settings. This review article will outline the challenges, which must be ameliorated prior to the widespread integration of multiplex immunoassays in clinical settings: (i) biomarker validation; (ii) standardisation of immunoassay design and quality control (calibration and quantification); (iii) availability, stability, specificity and cross-reactivity of reagents; (iv) assay automation and the use of validated algorithms for transformation of raw data into diagnostic results. A compendium of multiplex immunoassays applicable to in vitro diagnostics and a summary of the diagnostic products currently available commercially are included, along with an analysis of the relative states of development for each format (namely planar slide based, suspension and planar/microtitre plate based) with respect to the aforementioned issues

γδ T Cells Provide an Early Source of Interferon γ in Tumor Immunity

Interferon (IFN)-γ is necessary for tumor immunity, however, its initial cellular source is unknown. Because γδ T cells primarily produce this cytokine upon activation, we hypothesized that they would provide an important early source of IFN-γ in tumor immunosurveillance. To address this hypothesis, we first demonstrated that γδ T cell–deficient mice had a significantly higher incidence of tumor development after challenge with a chemical carcinogen methylcholanthrene (MCA) or inoculation with the melanoma cell line B16. In wild-type mice, γδ T cells were recruited to the site of tumor as early as day 3 after inoculation, followed by αβ T cells at day 5. We then used bone marrow chimeras and fetal liver reconstitutions to create mice with an intact γδ T cell repertoire but one that was specifically deficient in the capacity to produce IFN-γ. Such mice had a higher incidence of tumor development, induced either with MCA or by inoculation of B16 melanoma cells, compared with mice with IFN-γ–competent γδ T cells. Moreover, genetic deficiency of γδ T cells resulted in impaired IFN-γ production by tumor antigen-triggered αβ T cell upon immunization with tumor lysate. These results demonstrate that γδ T cells can play a necessary role in tumor immunity through provision of an early source of IFN-γ that in turn may regulate the function of tumor-triggered αβ T cells

Evaluating Markers for Treatment Selection Based on Survival Time

For many medical conditions there are several treatment options available to patients. We consider evaluating markers based on a simple treatment selection policy that incorporates information on the patient\u27s marker value exceeding a threshold. Although traditional regression methods may assess the effect of the marker and treatment on outcomes, it is appealing to quantify more directly the potential impact on the population of using the marker to select treatment. A useful tool is the selection impact (SI) curve proposed by Song and Pepe (2004, \textit{Biometrics} \textbf{60}, 874--883) for binary outcomes. However, this approach does not deal with continuous outcomes, nor does it adjust for other covariates that are important for treatment selection. In this paper, we propose the SI curve for general outcomes, with specific focus on the survival time. We further propose the covariate specific SI curve to incorporate covariate information in treatment selection. Nonparametric and semiparametric estimators are developed accordingly. We show that the proposed estimators are consistent and asymptotically normal. Simulation studies demonstrate that these estimators work well with realistic sample sizes. We illustrate the SI curve and the statistical inference for it with data from an AIDS clinical trial

Synthesis of honokiol analogues and evaluation of their modulating action on VEGF protein secretion and telomerase-related gene expressions

A group of 36 biphenyl derivatives structurally related to honokiol were synthesized by means of Suzuki coupling reactions. Their cytotoxicities were evaluated and compared to that of honokiol. Some of the compounds were then evaluated for their ability to downregulate the secretion of the VEGF protein and the expression of the VEGF, hTERT, and c-Myc genes; the two latter involved in the activation of telomerase in tumoral cells. Some of the synthetized derivatives showed promising pharmacological features as they exhibited IC50 values in low micromolar range, good therapeutic margins, and a multiple mode of action on tumor cells based on the inhibition of VEGF and, at the same time, of the expression of genes related to the activation of telomerase

Calcium and calcium sensing receptor modulates the expression of thymidylate synthase, NAD(P)H:quinone oxidoreductase 1 and survivin in human colon carcinoma cells: Promotion of cytotoxic response to mitomycin C and fluorouracil

Ca 2+ and the cell-surface calcium sensing receptor (CaSR) constitute a novel and robust ligand/receptor system in regulating the proliferation and differentiation of colonic epithelial cells. Here we show that activation of CaSR by extracellular Ca 2+ (or CaSR agonists) enhanced the sensitivity of human colon carcinoma cells to mitomycin C (MMC) and fluorouracil (5-FU). Activation of CaSR up-regulated the expression of MMC activating enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO-1) and down-regulated the expression of 5-FU target, thymidylate synthase (TS) and the anti-apoptotic protein survivin. Cells that were resistant to drugs expressed little or no CaSR but abundant amount of survivin. Disruption of CaSR expression by shRNA targeting the CaSR abrogated these modulating effects of CaSR activation on the expression of NQO1, TS, survivin and cytotoxic response to drugs. It is concluded that activation of CaSR can enhance colon cancer cell sensitivity to MMC and 5-FU and can modulate the expression of molecules involved in the cellular responses to these cytotoxic drugs. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61862/1/20470_ftp.pd

Non-stem cell lineages as an alternative origin of intestinal tumorigenesis in the context of inflammation

According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, has been shown to suppress intestinal stemness. Here, we used Paneth cells as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation in mice. Upon inflammation, Paneth cell-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in patients with inflammatory bowel disease, but also of a larger fraction of human sporadic colon cancers. The latter is possibly because of the inflammatory consequences of western-style dietary habits, a major colon cancer risk factor. Machine learning methods designed to predict the cell-of-origin of cancer from patient-derived tumor samples confirmed that, in a substantial fraction of sporadic cases, the origins of colon cancer reside in secretory lineages and not in stem cells.</p

Nervous-system-specific carcinogenesis by ethylnitrosourea in the rat: molecular and cellular aspects

A lead in the search for cellular determinants favoring neoplastic transformation may be provided by the pronounced tissue specificity of the oncogenic effect of certain carcinogens which do not require enzymatic metabolic activation, i.e., in cases where this specificity cannot be due to tissue differences in the activity of enzymes involved in the formation of the ultimate reactants. A carcinogen that fulfills this condition is the ethylating agent N-ethyl-N-nitrosourea (EtNU). Alkylation of nucleic acid constituents by N-nitroso compounds in relation to mutagenesis and carcinogenesis has received considerable attention recently

c-Myc overexpression sensitises colon cancer cells to camptothecin-induced apoptosis

The proto-oncogene c-Myc is overexpressed in 70% of colorectal tumours and can modulate proliferation and apoptosis after cytotoxic insult. Using an isogenic cell system, we demonstrate that c-Myc overexpression in colon carcinoma LoVo cells resulted in sensitisation to camptothecin-induced apoptosis, thus identifying c-Myc as a potential marker predicting response of colorectal tumour cells to camptothecin. Both camptothecin exposure and c-Myc overexpression in LoVo cells resulted in elevation of p53 protein levels, suggesting a role of p53 in the c-Myc-imposed sensitisation to the apoptotic effects of camptothecin. This was confirmed by the ability of PFT-alpha, a specific inhibitor of p53, to attenuate camptothecin-induced apoptosis. p53 can induce the expression of p21(Waf1/Cip1), an antiproliferative protein that can facilitate DNA repair and drug resistance. Importantly, although camptothecin treatment markedly increased p21(Waf1/Cip1) levels in parental LoVo cells, this effect was abrogated in c-Myc-overexpressing derivatives. Targeted inactivation of p21(Waf1/Cip1) in HCT116 colon cancer cells resulted in significantly increased levels of apoptosis following treatment with camptothecin, demonstrating the importance of p21(Waf1/Cip1) in the response to this agent. Finally, cDNA microarray analysis was used to identify genes that are modulated in expression by c-Myc upregulation that could serve as additional markers predicting response to camptothecin. Thirty-four sequences were altered in expression over four-fold in two isogenic c-Myc-overexpressing clones compared to parental LoVo cells. Moreover, the expression of 10 of these genes was confirmed to be significantly correlated with response to camptothecin in a panel of 30 colorectal cancer cell lines