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Intrinsic mitochondrial membrane potential and associated tumor phenotype are independent of MUC1 over-expression.

ABSTRACT: We have established previously that minor subpopulations of cells with stable differences in their intrinsic mitochondrial membrane potential (??m) exist within populations of mammary and colonic carcinoma cells and that these differences in ??m are linked to tumorigenic phenotypes consistent with increased probability of participating in tumor progression. However, the mechanism(s) involved in generating and maintaining stable differences in intrinsic ??m and how they are linked to phenotype are unclear. Because the mucin 1 (MUC1) oncoprotein is over-expressed in many cancers, with the cytoplasmic C-terminal fragment (MUC1 C-ter) and its integration into the outer mitochondrial membrane linked to tumorigenic phenotypes similar to those of cells with elevated intrinsic ??m, we investigated whether endogenous differences in MUC1 levels were linked to stable differences in intrinsic ??m and/or to the tumor phenotypes associated with the intrinsic ??m. We report that levels of MUC1 are significantly higher in subpopulations of cells with elevated intrinsic ??m derived from both mammary and colonic carcinoma cell lines. However, using siRNA we found that down-regulation of MUC1 failed to significantly affect either the intrinsic ??m or the tumor phenotypes associated with increased intrinsic ??m. Moreover, whereas pharmacologically mediated disruption of the ??m was accompanied by attenuation of tumor phenotype, it had no impact on MUC1 levels. Therefore, while MUC1 over-expression is associated with subpopulations of cells with elevated intrinsic ??m, it is not directly linked to the generation or maintenance of stable alterations in intrinsic ??m, or to intrinsic ??m associated tumor phenotypes. Since the ??m is the focus of chemotherapeutic strategies, these data have important clinical implications in regard to effectively targeting those cells within a tumor cell population that exhibit stable elevations in intrinsic ??m and are most likely to contribute to tumor progression.

SUBMITTER: Houston MA

PROVIDER: S-EPMC3179472 | biostudies-other | 2011

REPOSITORIES: biostudies-other

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Intrinsic mitochondrial membrane potential and associated tumor phenotype are independent of MUC1 over-expression.

Houston Michele A MA Augenlicht Leonard H LH Heerdt Barbara G BG

PloS one 20110923 9

We have established previously that minor subpopulations of cells with stable differences in their intrinsic mitochondrial membrane potential (Δψm) exist within populations of mammary and colonic carcinoma cells and that these differences in Δψm are linked to tumorigenic phenotypes consistent with increased probability of participating in tumor progression. However, the mechanism(s) involved in generating and maintaining stable differences in intrinsic Δψm and how they are linked to phenotype ar ...[more]

PMID: 21966455

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Project description:Non-destructive analysis of cells at the molecular level is of critical importance for cell research. At present, immunoassay-based and aptamer-based methods can achieve non-structural destructive cell analysis, but still lead to changes in cells at the molecular level. Here, we have proposed a dual-terminal amplification (DTA) strategy, which enables nondestructive analysis of membrane protein MUC1 without the effect on protein expression and cell viability in living cells. Methods: A fluorophore (Cy5)-labeled DNA ternary complex consisting of three oligonucleotides is designed. It can recognize MUC1 through its aptamer region, and thus make the MUC1 of cells visible under a fluorescence microscope. When DNA polymerase is added, dual-terminal amplification is performed. One direction dissociates aptamer from MUC1, and the other direction, also known as rolling circle amplification (RCA), produces long linear DNA strands, which can be further adopted for quantitative analysis of MUC1. In this way, all reagents are removed from the surface of the cells after the analysis, which allows nondestructive analysis. We named this strategy dual-terminal amplification (DTA) analysis. Results: By using the DTA analysis, both in situ fluorescence imaging analysis and ex situ fluorescence quantitative analysis of MUC1 were achieved. In addition, the aptamer-containing DNA ternary complex stays on cell surface only during the analysis and leaves the cell after the analysis is complete. The cells can be maintained in a non-interfering state for the rest of the time. So after the analysis, it is found that there are no effect on the physiological activity of cells and the expression of target protein even after two rounds of repeatable imaging and quantitative analysis. Conclusion: In summary, we have successfully constructed a strategy for nondestructive analysis of membrane protein in living cells. We believe that this method provides a promising way for the analysis of the key membrane proteins of cells and the versatile utilization of precious cell samples.

Dataset Information

Intrinsic mitochondrial membrane potential and associated tumor phenotype are independent of MUC1 over-expression.

Publications

Intrinsic mitochondrial membrane potential and associated tumor phenotype are independent of MUC1 over-expression.

Similar Datasets

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