Non Apoptogenic

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Non-apoptogenic Ca2+-Related

Extrusion of Mitochondriain Anoxia/Reoxygenation Stress

ANNALISA DORIO,a CLAUDIA CERELLA,a MILENA DE NICOLA,a

MARIA DALESSIO,a GIAMPIERO GUALANDI,b AND LINA GHIBELLIa

aDipartimento di Biologia, Universita di Roma Tor Vergata, 00133 Rome, Italy

bDABAC, Universita della Tuscia, 01011 Viterbo, Italy

ABSTRACT: Tumor cells often develop molecular strategies for survivalto anoxia/reoxygenation stress as part of tumor progression. Here wedescribe that the B lymphoma EpsteinBarr-positive cells E2r survivereoxygenation in spite of a very high and long-lasting increase in cytosolicCa2+ and the loss of about half of their mitochondria due to specificextrusion of the organelles from the cells. The extrusion typically occurs3 days after reoxygenation, and a regular mitochondrial asset is regainedafter further 24 h.

KEYWORDS: calcium; apoptosis; anoxia; reoxygenation; mitochondria

INTRODUCTION

Hypoxic/anoxic stress plays a major role in two of the most important hu-man pathologies, namely ischemia/reperfusion and tumors. On the one side,the rapid and strong blood flow stop occurring during accidental ischemiaabruptly causes severe anoxia, leading to cell death by apoptosis or necrosis,to an extent that depends on the duration/strength of ischemia; this producesirreversible damage to poorly renewable tissues (brain or cardiac). The majorcytotoxic phase occurs during reperfusion, when the reestablishment of tissuehomeostasis produces extensive cell death by apoptosis, the extent of whichstill depends on the gravity of the ischemic phase. This is accompanied by (andperhaps due to) an uncontrolled increase in cytosolic-free Ca2+ ([Ca2+]c), apotent inducer of apoptosis.

On the other side, during the growth of poorly vascularized tumor masses, ahypoxic environment partially impairs cell growth and viability, selecting cellsfor resistance to ischemic stress as part of tumor progression. Reoxygenation

Address for correspondence: Lina Ghibelli, Dipartimento di Biologia, Universita di Roma Tor Ver-gata, via Ricerca Scientifica 1, 00133 Roma, Italy. Voice:+39-06-7259-4323; fax:+39-06-2023500.

[email protected]

Ann. N.Y. Acad. Sci. 1099: 512515 (2007). C 2007 New York Academy of Sciences.

doi: 10.1196/annals.1387.067

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is gradually provided by neoangiogenesis; in culture this is a nontoxic process,

suggesting that tumor cells have developed strategies for survival to reoxy-

genation.Thus, return to homeostasis is more cytotoxic for nerve/cardiac cells after

ischemia, than for tumor cells. Much effort is being posed in envisaging ther-

apeutic protocols to get the reverse. Might we learn lessons from tumor cellsstrategies for survival?

MATERIALS AND METHODS

Cells: E2r are BL41 human B lymphoma cells converted by in vitro infectionwith EpsteinBarr virus.1 Anoxia was provided by culturing cells in tissueculture flasks placed in an atmosbag (from Sigma, St. Louis, MO) filled with a

controlled mixture of 5% CO2; 95% N2 (nominal anoxia). For reoxygenation,

cells were placed in a regular CO2 incubator. Mitochondria were labeled with

mitotracker red, and visualized by a fluorescent microscope.2 Cell viability andapoptosis were assessed by a fluorescent microscopy analysis of cells labeled

with the cell-impermeant dye iodidium propide (viable cell are not stained)

and with the cell-permeant dye Hoechst, which allow recognition of apoptotic

cells, respectively.3

Cytosolic Ca2+

concentration was assessed by loadingcells with the specific dye Fluo3-AM; fluorescence was then quantified byflow cytometric analysis.4 .

RESULTS AND DISCUSSION

During nominal anoxia, E2r cells suffer a slight cytotoxicity in terms ofapoptosis and a drastic decrease in the proliferation rate (not shown). Upon

reoxygenation, apoptosis is no longer detectable over the basal values foundin control cultures, and the duplication rate is soon recovered, as shown in

FIGURE 1 A. Thus, E2r fully and immediately recover in terms of viability and

proliferation. To understand if this lack of cytotoxicity is due to the containmentof [Ca2+]c or it occurs despite it, a time course of [Ca2+]c was performed at

increasing times of reoxygenation. As shown in FIGURE 1B,[Ca2+]c concentra-

tion very rapidly rises to values that are far beyond those normally compatible

with cell survival (almost 2 mM); the extra Ca2+ is partially cleared at 24 h and

48 h, to return at normal levels at 72 h. Since promotion of [Ca 2+]c increase is

a major cytotoxic mechanism of reoxygenation, this indicates that survival toreoxygenation by E2r occurs in spite of high [Ca2+]c, and implies mechanisms

that act downstream to it. Interestingly, a surprising phenomenon accompanies

the recovery to normal [Ca2+]c, i.e., the extrusion of mitochondria. The image

of a E2r cell in the process of extruding mitochondria is shown in FIGURE 1C


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FIGURE 1. Cell proliferation and apoptosis in E2r cells during reoxygenation. Every

day 500,000 cells were plated and counted after 24 h (A); average of four independent exper-

iments SD. Cytosolic Ca2+ concentration ([Ca2+]c) was measured at the indicated time

points as described in Materials and Methods; data are the average of four independent

experiments SD (B). Panel C shows the mitochondrial pattern of untreated (top) versus

reoxygenated (72 h, bottom) E2r cells upon specific mitochondria labeling (see Materialsand Methods).

(bottom), compared with an untreated cell (top). The normal pattern of mi-

tochondria unevenly distributed in the cytoplasm, changes to a pattern where

mitochondria group into several (see picture) or unique (not shown) masses,to be released in the extracellular space. The amount of the released mitochon-

dria is about half of the total cell mass; a normal amount is readily recovered

after further 24 h. No loss of cell viability accompanies the phenomenon. To

our knowledge, such a phenomenon was never described, and we are actively

working in order to uncover the mechanisms through which this occurs, and the

role it may play in the survival of tumor cells to anoxia/reoxygenation. Indeed,it will be important to determine whether cells survive in spite of mitochondria

loss or due to it: since mitochondrial extrusion coincides with the recovery of

normal [Ca2+]c, it is tempting to speculate that mitochondria extrusion is a

mean to dispose of the extra Ca2+ accumulated during reoxygenation.


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REFERENCES

1. DALESSIO,M.,M. DENICOLA, S . COPPOLA, etal. 2005. Oxidative Bax dimerizationpromotes its translocation to mitochondria independently of apoptosis. FASEBJ. 19: 15041506.

2. DENICOLA, M . , G . GUALUANDI, A . ALFONSI, et al. 2006. Different fates of intracel-lular glutathione determine different modalities of apoptotic nuclear vesiculation.Biochem. Pharmacol. 72: 14051416.

3. FANELLI, C., S. COPPOLA, R. BARONE, et al. 1999. Magnetic fields increase cellsurvival by inhibiting apoptosis via modulation of Ca2+ influx. FASEB J. 13:95102.

4. CERELLA, C . , M . DAlessio, M. DENICOLA, et al. 2003. Cytosolic and endoplasmicreticulum Ca2+ concentrations determine the extent and the morphological typeof apoptosis, respectively. Ann. N. Y. Acad. Sci. 1010: 7477.

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