Elsevier

Mitochondrion

Volume 13, Issue 6, November 2013, Pages 662-667
Mitochondrion

A bioenergetic profile of non-transformed fibroblasts uncovers a link between death-resistance and enhanced spare respiratory capacity

https://doi.org/10.1016/j.mito.2013.09.005Get rights and content

Highlights

  • We used human cells that acquired apoptosis resistance after a single genotoxic exposure.

  • The intrinsic bioenergetic profile of these death-resistant cells was investigated.

  • Death-resistant cells displayed enhanced mitochondrial spare respiratory capacity (SRC).

  • Enhanced SRC may represent an early phenotypic alteration in neoplastic transformation.

Abstract

Apoptosis-resistance and metabolic imbalances are prominent features of cancer cells. We have recently reported on populations of human fibroblasts that exhibit resistance to mitochondrial-mediated apoptosis, acquired as a result of a single genotoxic exposure. The objective of the present study was to investigate the intrinsic bioenergetic profile of the death-resistant cells, as compared to the clonogenic control cells. Therefore, we analyzed the basic bioenergetic parameters including oxygen consumption and extracellular acidification rates, coupling efficiency, and spare respiratory capacity. Our data demonstrate a strong correlation between enhanced spare respiratory capacity and death-resistance, which we postulate to be indicative of the earliest stages of carcinogenesis.

Introduction

Several lines of evidence have indicated that carcinogenesis evolves from consecutive genetic and/or epigenetic alterations that provide cellular survival advantages, ultimately leading to the conversion of normal human cells to malignant cancer cells [for review, see (Hanahan and Weinberg, 2000, Hanahan and Weinberg, 2011)]. As evasion of apoptosis and unlimited replicative potential are hallmarks of cancer, two questions become inevitable when studying death-resistance and early-stage carcinogenesis: 1) how does a cell gain enhanced proliferative capacity, and 2) what fuels this unlimited replicative potential? In most cells under aerobic conditions, glucose is converted to pyruvate through glycolysis, which then enters the TCA cycle where the flavin nucleotide (FADH2) and NADH are produced. The respective reduced equivalents are further oxidized by the mitochondrially-localized electron transport chain (ETC), which ultimately produces ~ 80% of total cellular ATP. Conversely, early studies in tumor cells have found an upregulation of glycolysis for energy production, even in the presence of sufficient oxygen levels. This phenotype is known as the Warburg effect, after Dr. Otto Warburg who uncovered the phenomenon (Warburg, 1956). While the high glycolytic phenotype of cancer cells has been exploited for clinical use (i.e. positron emission tomography), the molecular basis of the Warburg effect remains unclear.

The bioenergetic profile of several cell types such as neurons, endothelial cells, and human carcinoma cell lines, has been characterized in an effort to uncover alterations that may be targeted for therapeutic purposes (Rodriguez-Enriquez et al., 2008, van der Windt et al., 2012, Wu et al., 2007, Xun et al., 2012). Of particular interest to the present study, is the role that spare respiratory capacity (SRC) may play in death resistance. The term, SRC, describes the reserve capacity that enables the production of energy in response to cellular stress (Nicholls, 2009). It has been hypothesized that in the face of oxidative stress, cell survival can be potentiated when a maximal reserve of ATP is maintained (Choi et al., 2009, Fern, 2003, Zhu et al., 2012).

We have previously generated sub-populations of BJ-hTERT human diploid foreskin fibroblasts, which have acquired resistance to cell death induced by hexavalent chromium [Cr(VI)], a broad-spectrum DNA-damaging agent (Pritchard et al., 2005). Fibroblasts are integral to the cellular microenvironment and have been associated with pathological conditions such as fibrosis and carcinogenesis (McAnulty, 2007, Vaheri et al., 2009). This system is unique in that it models initial molecular events that occur in a normal cell that survived a single, acute, initiating genotoxic challenge. While the selection model in this study was generated by Cr(VI) treatment, it also exhibits a cross-resistance to the well-known chemotherapeutic agent, cisplatin, as well as to H2O2 (Nickens et al., 2012). Long-term exposure to certain forms of Cr(VI) is associated with respiratory carcinogenesis (IARC, 1990). Our recent report investigated the death-sensitivity of subclonal populations derived from clonogenic survivors of BJ-hTERT cells treated with Cr(VI) (DR), or selected by dilution-based cloning without treatment (CC) (Nickens et al., 2012). Notably, our data suggested the presence of more resilient mitochondria in DR cells, and that death resistance can be acquired in normal human cells early after genotoxin exposure. Taken together, these data led us to postulate that resistance to mitochondrially-mediated cell death and mitochondrial dysregulation may be initial phenotypic alterations associated with early-stage carcinogenesis.

Here we report on the bioenergetic profile of the DR and CC subclonal cell lines. By employing the Seahorse Bioscience XF Extracellular Flux Analyzer, we simultaneously measured glycolysis by assessing the extracellular acidification rate (ECAR), as well as the rate of oxidative phosphorylation by measuring the cellular oxygen consumption rate (OCR) (Eklund et al., 2004). We tested the hypothesis that survival after genotoxic stress may involve the selection of cells with intrinsically altered bioenergetic regulation. Our data show that while there is no difference in basal ATP content, ECAR, or OCR, there is an increase in the SRC of the DR cells, as compared to the CC cells. Taken together, the present data show that a greater intrinsic SRC is coincident with death-resistance in our model system. Moreover, this enhanced capacity may be a mechanistic step in the acquisition of death-resistance, which in turn may potentially foster neoplastic progression. Importantly, we show that the intrinsically enhanced SRC was observed in diploid human cells that have acquired a death-resistant phenotype following only a single exposure to a carcinogen.

Section snippets

Subcloning, cell lines, and culture parameters

Subclonal populations were derived as previously described (Nickens et al., 2012). The cell lines used in the present study include untreated clonogenic control cell lines, CC1 and CC2, as well as clones derived from clonogenic survivors of Cr(VI) exposure, DR1, DR2, DR3, and DR4, which display an apoptosis resistant phenotype. As previously reported, all CC and DR cell lines were derived from human foreskin fibroblasts transfected with the hTERT gene (BJ-hTERT; Geron Corp.), and further

Results and discussion

Two death-resistant cell lines (DR1, DR2), as well as a death-susceptible clonogenic control line (CC1) were the focus of a recently published report from our laboratory (Nickens et al., 2012). Our data showed that, as compared to the death-susceptible cells, the DR cells were resistant to apoptosis as indicated by lack of cleaved caspase 3 expression following exposure to both Cr(VI) and cisplatin. The mitochondrial-mediated pathway of apoptosis was inhibited in the DR cell, as indicated by

Acknowledgments

The authors would like to acknowledge Dr. Eric Kaldjian for his assistance in this collaborative effort, as well as Dr. Travis O'Brien, Dr. Madhu Lal-Nag, and Dr. Tura Camilli for their helpful discussions and insight. This work was supported by the National Institutes of Health [R21ES017334 and R01CA107972 to S.C., R01CA107972 supplement to K.N., and R01ES05304 and R01ES09961 to S.P.].

References (38)

  • X.L. Zu et al.

    Cancer metabolism: facts, fantasy, and fiction

    Biochem. Biophys. Res. Commun.

    (2004)
  • B.H. Ahn et al.

    A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis

    Proc. Natl. Acad. Sci. U. S. A.

    (2008)
  • N. Bellance et al.

    Mitochondria: from bioenergetics to the metabolic regulation of carcinogenesis

    Front. Biosci.

    (2009)
  • A.G. Bodnar et al.

    Extension of life-span by introduction of telomerase into normal human cells

    Science

    (1998)
  • M.D. Brand et al.

    Assessing mitochondrial dysfunction in cells

    Biochem. J.

    (2011)
  • C.A. Caneba et al.

    Pyruvate uptake is increased in highly invasive ovarian cancer cells under anoikis conditions for anaplerosis, mitochondrial function, and migration

    Am. J. Physiol. Endocrinol. Metab.

    (2012)
  • S.W. Choi et al.

    Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure

    J. Neurochem.

    (2009)
  • S.E. Eklund et al.

    A microphysiometer for simultaneous measurement of changes in extracellular glucose, lactate, oxygen, and acidification rate

    Anal. Chem.

    (2004)
  • R. Fern

    Variations in spare electron transport chain capacity: the answer to an old riddle?

    J. Neurosci. Res.

    (2003)
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    This work was conducted in partial fulfillment of the requirements of the Ph.D. degree in Molecular Medicine, Columbian College of Arts and Sciences, The George Washington University.

    1

    Current addresses: KPN, Center for Prostate Disease Research, 1530 East Jefferson Street, Rockville, MD 20852, United States; SRP, Duke Cancer Institute, Box 3917, Seeley Mudd Building, Room 412, 2nd Floor, 10 Bryan Searle Drive, Durham, NC 27710, United States.

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